Syed S. Islam

Bladder tissue engineering: Tissue regeneration and neovascularization of HA-VEGF-incorporated bladder acellular constructs in mouse and porcine animal models

Successful tissue engineering requires appropriate recellularization and vascularization. Herein, we assessed the regenerative and angiogenic effects of porcine bladder acellular matrix (ACM) incorporated with hyaluronic acid (HA) and vascular endothelial growth factor (VEGF) in mouse and porcine models. Prepared HA-ACMs were rehydrated in different concentrations of VEGF (1, 2, 3, 10, and 50 ng/g ACM). Grafts were implanted in mice peritoneum in situ for 1 week. Angiogenesis was quantified with CD31 and Factor VIII immunostaining using Simple PCI. Selected optimal VEGF concentration that induced maximum vascularization was then used in porcine bladder augmentation model. Implants were left in for 4 and 10 weeks. Three groups of six pigs each were implanted with ACM alone, HA-ACM, and HA-VEGF-ACM. Histological, immunohistochemical (Uroplakin III, alpha-SMA, Factor VIII), and immunofluorescence (CD31) analysis were performed to assess graft regenerative capacity and angiogenesis. In mouse model, statistically significant increase in microvascular density was demonstrated in the 2 ng/g ACM group. When this concentration was used in porcine model, recellularization increased significantly from weeks 4 to 10 in HA-VEGF-ACM, with progressive decrease in fibrosis. Significantly increased vascularization, coupled with increased urothelium and smooth muscle cell (SMC) regeneration, was observed in HA-VEGF grafts at week 10 in the center and periphery, compared with week 4. HA-VEGF grafts displayed highest in vivo epithelialization, neovascularization, and SMCs regeneration. A total of 2 ng/g tissue VEGF when incorporated with HA proved effective in stimulating robust graft recellularization and vascularization, coordinated with increased urothelial bladder development and SMC augmentation into bundles by week 10.

Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer

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.

TGF-β1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner

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.

Urinary bladder tissue engineering using natural scaffolds in a porcine model: role of Toll-like receptors and impact of biomimetic molecules.

Introduction: Natural scaffolds have been shown to induce T helper 2 (TH2)-specific immune responses in host tissues; however, the precise mechanisms that underlie this immune response are unknown. Using a porcine animal model, we evaluated the role of Toll-like receptors (TLRs) and matrix remodelling in the implantation of bladder acellular matrix (ACM) grafts and ACMs fortified with biomimetic materials. Materials and Methods: Bladders were decellularized with detergent and treated in 3 different ways prior to implantation: ACM alone, hyaluronic acid (HA)-ACM and HA-vascular endothelial growth factor (VEGF)-ACM. Animals were sacrificed at 4 or 10 weeks post-implantation and total gene expressions for TH2 (IL-4), TH1 (IFN-γ), TLR2, TLR4, and TGF-β1 were analyzed using real-time RT-PCR. Using histology (H&E and Masson’s trichrome) and immunohistochemistry (uroplakin, α-smooth muscle actin, CD31 and factor VIII) the regenerative capacity was correlated with the gene expression of different proteins. Results: IL-4, TLR2, and TLR4 gene expression were markedly decreased at 4 and 10 weeks in both the HA-ACM group and the HA-VEGF-ACM group compared to ACM alone. IFN-γ expression was negligible in all groups and time periods. TGF-β1 expression was highest in the HA- and VEGF-treated grafts. Recellularization was inversely proportional to TLR and TH2 expression but proportional to TGF-β1. Conclusion: ACM alone grafts demonstrated stronger TLR4 expression which may promote a distinct TH2 immune response and a reduced regenerative capacity in grafts. Treatment of grafts with HA and VEGF may help regulate host immune responses by reducing TLR4 and IL-4 and increasing TGF-β1.

Combination of carbonic anhydrase inhibitor, acetazolamide, and sulforaphane, reduces the viability and growth of bronchial carcinoid cell lines

BackgroundBronchial carcinoids are pulmonary neuroendocrine cell-derived tumors comprising typical (TC) and atypical (AC) malignant phenotypes. The 5-year survival rate in metastatic carcinoid, despite multiple current therapies, is 14-25%. Hence, we are testing novel therapies that can affect the proliferation and survival of bronchial carcinoids.MethodsIn vitro studies were used for the dose–response (AlamarBlue) effects of acetazolamide (AZ) and sulforaphane (SFN) on clonogenicity, serotonin-induced growth effect and serotonin content (LC-MS) on H-727 (TC) and H-720 (AC) bronchial carcinoid cell lines and their derived NOD/SCID mice subcutaneous xenografts. Tumor ultra structure was studied by electron microscopy. Invasive fraction of the tumors was determined by matrigel invasion assay. Immunohistochemistry was conducted to study the effect of treatment(s) on proliferation (Ki67, phospho histone-H3) and neuroendocrine phenotype (chromogranin-A, tryptophan hydroxylase).ResultsBoth compounds significantly reduced cell viability and colony formation in a dose-dependent manner (0–80 μM, 48 hours and 7 days) in H-727 and H-720 cell lines. Treatment of H-727 and H-720 subcutaneous xenografts in NOD/SCID mice with the combination of AZ + SFN for two weeks demonstrated highly significant growth inhibition and reduction of 5-HT content and reduced the invasive capacity of H-727 tumor cells. In terms of the tumor ultra structure, a marked reduction in secretory vesicles correlated with the decrease in 5-HT content.ConclusionsThe combination of AZ and SFN was more effective than either single agent. Since the effective doses are well within clinical range and bioavailability, our results suggest a potential new therapeutic strategy for the treatment of bronchial carcinoids.

Impact of bladder-derived acellular matrix, growth factors, and extracellular matrix constituents on the survival and multipotency of marrow-derived mesenchymal stem cells.

We investigate the effect of bladder-derived acellular matrix (ACM) on bone marrow mesenchymal stem cells (BM-MSC) growth, survival, and differentiation, and evaluate the effect of collagen I and IV on BM-MSC differentiation potential to SMC. BM-MSCs isolated from CD1(_) mice were characterized by surface markers and differentiation into different lineages. BM-MSC SMC potential was further evaluated in stem cell medium alone or supplemented with TGF-β1 and recombinant human platelet-derived growth factor (PDGF-BB) on plastic, collagen I and IV using western blot. Furthermore, BM-MSCs were seeded on porcine derived ACM-fortified with hyaluronic acid and cultured in Mesencult+-growth factors, bone, or fat induction media for 3 weeks. Seeded constructs were evaluated by H&E, Ki67 assay, Oil red O, and Alizarin red stain. SMC differentiation was semiquantified via immunohistochemistry. BM-MSCs differentiated into fat and bone when induced. In Mesencult, BM-MSCs differentiated into SMC, expressing α-SMA, calponin, and MHC. BM-MSCs cultured on collagen I and IV reduced expression of SMC and MHC compared to plastic. On ACM-HA, BM-MSCs maintained multipotent state by differentiating to bone and fat when induced. In Mesencult, BM-MSC-seeded ACM-HA expressed α-SMA, calponin, and MHC. TGF-β1 and PDGF-BB enhanced SMC differentiation on collagens and ACM-HA. SMC proteins expression by BM-MSC varies depending on culture substrate. SMC markers are expressed higher on plastic and lower on collagen I, IV, and ACM-HA, suggesting these substrates preferentially maintain undifferentiated state of BM-MSC, which could be advantageous for incorporation of cell-seeded grafts to permit host modulation of tissue regeneration.

Simultaneous Targeting of Bladder Tumor Growth, Survival, and Epithelial-to-Mesenchymal Transition with a Novel Therapeutic Combination of Acetazolamide (AZ) and Sulforaphane (SFN).

BackgroundCurrent chemotherapies for advanced stage metastatic bladder cancer often result in severe side effects, and most patients become drug resistant over time. Thus, there is a need for more effective therapies with minimal side effects.ObjectiveThe acid/base balance in tumor cells is essential for tumor cell functioning. We reasoned that simultaneous targeting of pH homeostasis and survival pathways would improve therapeutic efficacy. We evaluated the effectiveness of targeting pH homeostasis with the carbonic anhydrase inhibitor acetazolamide (AZ) in combination with the survival pathway targeting isothiocyanate sulforaphane (SFN) on the HTB-9 and RT112(H) human bladder tumor cell lines.Materials and MethodsWe assessed viability, proliferation, and survival in vitro and effect on xenografts in vivo.ResultsCombination AZ + SFN treatment induced dose-dependent suppression of growth, produced a potent anti-proliferative and anti-clonogenic effect, and induced apoptosis through caspase-3 and PARP activation. The anti-proliferative effect was corroborated by significant reductions in Ki-67, pHH3, cyclin D1, and sustained induction of the cell cycle inhibitors, p21 and p27. Both active p-Akt (Ser473) and p-S6 were significantly downregulated in the AZ + SFN combination treated cells with a concomitant inhibition of Akt kinase activity. The inhibitory effects of the AZ + SFN combination treatment showed similar efficacy as the dual PI3K/mTOR pathway inhibitor NVP-BEZ235, albeit at an expected higher dose. In terms of the effect on the metastatic potential of these bladder cancers, we found downregulated expression of carbonic anhydrase 9 (CA9) concomitant with reductions in both E-cadherin, N-cadherin, and vimentin proteins mitigating the epithelial-to-mesenchymal transition (EMT), suggesting negation of this program.ConclusionWe suggest that reductions in these components could be linked with downregulation of the survival mediated Akt pathway and suggested an active role of the Akt pathway in bladder cancer. Altogether, our in vitro and pre-clinical model data support the potential use of an AZ + SFN combination for the treatment of bladder cancer.

Spatio-Temporal Distribution of Smads and Role of Smads/TGF-β/BMP-4 in the Regulation of Mouse Bladder Organogenesis

Although Shh, TGF-β and BMP-4 regulate radial patterning of the bladder mesenchyme and smooth muscle differentiation, it is not known what transcription factors, local environmental cues or signaling cascades mediate bladder smooth muscle differentiation. We investigated the expression patterns of signaling mediated by Smad2 and Smad3 in the mouse embryonic bladder from E12.5 to E16.5 by using qRT-PCR, in situ hybridization and antibodies specifically recognizing individual Smad proteins. The role of Smad2 and Smad3 during smooth muscle formation was examined by disrupting the Smad2/3 signaling pathway using TβR1 inhibitor SB-431542 in organ culture system. qRT-PCR results showed that R-Smads, Co-Smad and I-Smads were all expressed during bladder development. RNA ISH for BMP-4 and immunostaining of TGF-β1 showed that BMP-4 and TGF-β1 were expressed in the transitional epithelium, lamina propia and muscularis mucosa. Smad1, Smad5 and Smad8 were first expressed in the bladder epithelium and continued to be expressed in the transitional epithelium, muscularis mesenchyme and lamina propia as the bladder developed. Smad2, Smad3 and Smad4 were first detected in the bladder epithelium and subsequently were expressed in the muscularis mesenchyme and lamina propia. Smad6 and Smad7 showed overlapping expression with R-Smads, which are critical for bladder development. In bladder explants (E12.5 to E16.5) culture, Smad2 and Smad3 were found localized within the nuclei, suggesting critical transcriptional regulatory effects during bladder development. E12.5 to E16.5 bladders were cultured with and without TβR1 inhibitor SB-431542 and assessed by qRT-PCR and immunofluorescence. After three days in culture in SB-431542, α-SMA, Smad2 and Smad3 expressions were significantly decreased compared with controls, however, with no significant changes in the expression of smooth muscle myosin heavy chain (SM-Myh. Based on the Smad expression patterns, we suggest that individual or combinations of Smads may be necessary during mouse bladder organogenesis and may be critical mediators for bladder smooth muscle differentiation.

Eugenol potentiates cisplatin anti-cancer activity through inhibition of ALDH-positive breast cancer stem cells and the NF-κB signaling pathway†

Triple‐negative breast tumors are very aggressive and contain relatively high proportion of cancer stem cells, and are resistant to chemotherapeutic drugs including cisplatin. To overcome these limitations, we combined eugenol, a natural polyphenolic molecule, with cisplatin to normalize cisplatin mediated toxicity and potential drug resistance. Interestingly, the combination treatment provided significantly greater cytotoxic and pro‐apoptotic effects as compared to treatment with eugenol or cisplatin alone on several triple‐negative breast cancer cells both in vitro and in vivo. Furthermore, adding eugenol to cisplatin potentiated the inhibition of breast cancer stem cells by inhibiting ALDH enzyme activity and ALDH‐positive tumor initiating cells. We provide also clear evidence that eugenol potentiates cisplatin inhibition of the NF‐κB signaling pathway. Indeed, the binding of NF‐κB to its cognate binding sites present in the promoters of IL‐6 and IL‐8 was dramatically reduced, which led to potent down‐regulation of the IL‐6 and IL‐8 cytokines upon combination treatment relative to the single agents. Similar effects were observed on proliferation, inhibition of epithelial‐to‐mesenchymal transition and stemness markers in tumor xenografts. These results provide strong preclinical justification for combining cisplatin with eugenol as therapeutic approach for triple‐negative breast cancers through targeting the resistant ALDH‐positive cells and inhibiting the NF‐κB pathway.

Abstract 2907: Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway

Triple-negative subtype of breast cancer (TNBC) contains exclusively tumorigenic and often endowed with self-renewing and resistance to cytotoxic chemotherapy, such as, paclitaxel resulting in relative increases in CSCs phenotype. Signaling through NF-kB may be essential for the CSCs self-renewal and could present potential target for novel treatment in breast cancer patients. We explored the possible involvement of NF-kB pathway that contribute to the functions and maintenance of breast CSCs and targeting this pathway by cisplatin combining with a natural product eugenol both in vitro and in vivo, using estrogen dependent (MCF-7) and independent (MDA-MB-231, MDA-MB-468 and BT-20) cell lines and xenografted both subcutaneous and orthotopic (fat pad) sites in nude mice. Chromatin immunoprecipitation (ChIP) assay was utilized to assess the NF-kB associated DNA binding capacity in promoter region. Surprisingly, neither cisplatin nor eugenol was capable of complete diminishing the CSCs populations. Cisplatin alone administration partially diminished apoptosis, mammospheres formation, aldehyde dehydrogenase 1 (ALDH1) activity, invasion and growth on nude mice. We observed cisplatin-induced activation of NF-kB associated with survival and regrowth of mamospheres. For effective elimination of CSCs phenotype by cisplatin, we cotreated cells with eugenol. Combination of eugenol and cisplatin significantly eradicated cisplatin induced NF-kB DNA binding capability assed by EMSA and ChIP assays, which was associated with abrogated apoptosis, mammospheres formation, ALDH1 activity and invasion. In vivo, combination therapy reduced the tumor size in synergistic manner. This was possibly due to induction of apoptosis, inhibition of proliferation, angiogenesis and downregulation of cisplatin-induced expression of proteins of NF-kB target genes. Although treatment of mammospheres by cisplatin partially induced the CSCs population through interleukin (IL)-6 pathway, however, only coadministration of eugenol and cisplatin reduced the numbers of CSCs to virtually undeletable levels in vitro and in vivo. Our data suggest that eugenol may be well suited to increase targeting of breast CSCs by cisplatin. Citation Format: Syed S. Islam, Al-Sharif Ibtehaj, Sultan Ahlam, Abdelilah Aboussekhra. Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2907.