Fistula-Associated Anal Adenocarcinoma: A 20-Year Single-Center Experience.

BACKGROUND: Fistula-associated anal adenocarcinoma (FAAC) is a rare consequence in patients with long-standing perianal fistulas. A paucity of data are available for this patient collective, making clinical characterization and management of this disease difficult. OBJECTIVE: This study aimed to describe a single-center experience with FAAC patients, their clinical course, and histopathological and molecular pathological characterization. METHODS: All patients receiving surgery for an anal fistula in 1999-2019 at a tertiary university referral hospital were included in this retrospective analysis. Patients with FAAC were eligible for histopathological analysis, including immunohistochemistry and molecular profiling. RESULTS: This study included 1004 patients receiving surgical treatment for an anal fistula, of whom 242 had an underlying inflammatory bowel disease (IBD). Ten patients were diagnosed with a fistula-associated anal carcinoma (1.0%), and six of these patients had an FAAC (0.6%). The mean overall survival of FAAC patients was 24 ± 3 months. FAAC immunohistochemistry revealed positive staining for CK20, CDX2 and MUC2, while stainings for CK5/6 and CK7 were negative. All FAAC specimens revealed microsatellite stability. Molecular profiling detected mutations in 35 genes, with the most frequent mutations being TP53, NOTCH1, NOTCH3, ATM, PIK3R1 and SMAD4. CONCLUSION: FAAC is rare but associated with poor clinical outcome. Tissue acquisition is crucial for early diagnosis and therapy and should be performed in long-standing, non-healing, IBD-associated fistulas in particular. The immunophenotype of FAAC seems more similar to the rectal-type mucosa than the anal glands.

Methylated Xanthones from the Rootlets of Metaxya rostrata Display Cytotoxic Activity in Colorectal Cancer Cells.

The tree fern Metaxya rostrata (Kunth) C. Presl is common in the rainforests of Central and South America, where suspensions of the dried rhizome are traditionally used to treat intestinal diseases. Two compounds from this plant, 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB), have been shown to be biologically highly active against colorectal cancer (CRC) cells in previous studies. The current investigation resulted in the isolation of the previously undescribed methylated xanthones 2-deprenyl-6-O-methyl-7-hydroxy-rheediaxanthone B, 2-deprenyl-5-O-methyl-7-methoxy-rheediaxanthone B, 2-deprenyl-5-O-methyl- 7-hydroxy-rheediaxanthone B and 2-deprenyl-7-methoxy-rheediaxanthone B. All compounds were isolated by column chromatography, structures were elucidated by one- and two-dimensional NMR-experiments and the identities of the compounds were confirmed by LC-HRMS. In logarithmically growing SW480 CRC cell cultures, cytotoxicity by neutral red uptake and MTT assays as well as caspase activation was analyzed. Cellular targets were examined by Western blot, and topoisomerase I (topo I) inhibition potential was tested. Comparing the structure-activity relationship with XB and OH-XB, the monomethylated derivatives showed qualitatively similar effects/mechanisms to their nonmethylated analogues, while dimethylation almost abolished the activity. Inhibition of topo I was dependent on the presence of an unmethylated 7-OH group.

Flavonoids Distinctly Stabilize Lymph Endothelial- or Blood Endothelial Disintegration Induced by Colon Cancer Spheroids SW620.

The health effects of plant phenolics in vegetables and other food and the increasing evidence of the preventive potential of flavonoids in "Western Diseases" such as cancer, neurodegenerative diseases and others, have gained enormous interest. This prompted us to investigate the effects of 20 different flavonoids of the groups of flavones, flavonols and flavanones in 3D in vitro systems to determine their ability to inhibit the formation of circular chemorepellent induced defects (CCIDs) in monolayers of lymph- or blood-endothelial cells (LECs, BECs; respectively) by 12(S)-HETE, which is secreted by SW620 colon cancer spheroids. Several compounds reduced the spheroid-induced defects of the endothelial barriers. In the SW620/LEC model, apigenin and luteolin were most active and acacetin, nepetin, wogonin, pinocembrin, chrysin and hispidulin showed weak effects. In the SW620/BEC model acacetin, apigenin, luteolin, wogonin, hispidulin and chrysin exhibited weak activity.

Destabilization of FoxM1 and Inhibition of Topoisomerase I Contribute to Cytotoxicity of Prenylated Xanthones Isolated from Metaxya rostrata.

We recently isolated the prenylated xanthones 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB) from the South American tree fern Metaxya rostrata. This study explores the mechanisms underlying the FoxM1 downregulation induced by both xanthones. Analysis of cell viability and cell-death induction in SW480, HCT116, Caco-2, DLD1 and HT29 exposed to xanthones found cell-loss and activation of caspase in all cell lines except HT29 that do not have high FoxM1 protein levels. To determine the cellular mechanism of xanthone-induced FoxM1 loss, protein stability was analyzed by cycloheximide-chase experiments and showed reduction of FoxM1 stability by XB but not OH-XB. Destabilization was prevented by inhibiting proteasome activity using MG-132 and moderately by the lysosomal inhibitor bafilomycin A1 (baf A1). OH-XB had a stronger impact than XB on FoxM1 mRNA expression by qRT-PCR, and MG-132 positively affected FoxM1 protein level in OH-XB exposed cells even though no decrease in protein abundance had been induced by the xanthone. Additionally, the compound inhibited topoisomerase I causing DNA DSB and early cell cycle arrest. This may reduce FoxM1 gene expression, which may in turn compromise DNA repair and enhance xanthone-induced cell death. With regard to xanthone-induced cell death, MG-132 protected cultures from cell loss induced by both compounds, and baf A1 was active against these XB-induced effects. In summary, both destabilization of FoxM1 protein and topoisomerase I inhibition contribute to both XB and OH-XB cytotoxic activity albeit at different ratios.

Prenylated xanthones from Metaxya rostrata suppress FoxM1 and induce active cell death by distinct mechanisms.

BACKGROUND: Metaxya rostrata C.Presl (Metaxyaceae) is a tree fern widespread in Central and South America and the dried rhizome is used in ethnic medicine against intestinal ulcers or tumors. An activity-guided isolation resulted in two structurally related xanthones: 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB). HYPOTHESIS/PURPOSE: This study analyzed the cytotoxic activity and underlying cellular mechanisms of OH-XB for the first time in comparison to XB. METHODS: We exposed the colorectal cancer cell line SW480 and F331 fibroblasts to XB and OH-XB and determined cell viability by neutral red uptake and nuclear morphology by staining with Hoechst dye. Cell cycle distribution and the mechanism of cell death were analyzed by FACS and western blot. Knockdown of FoxM1 expression was performed with siRNA. RESULTS: OH-XB was at least as cytotoxic as XB in the induction of cell cycle arrest and active cell death. While both compounds strongly inhibited the transcription factor FoxM1, the cellular mechanisms of growth arrest and cell death induction differed widely: OH-XB induced S-phase cell cycle arrest in contrast to a G2-M-phase arrest by XB. It caused morphological modifications typical for classical apoptosis with increased caspase 7 activity and enhanced cleavage of PARP, while XB caused caspase 2 activation and mitotic catastrophe. After knockdown of FoxM1 expression no induction of caspase activity could be observed. CONCLUSION: In summary, our data clearly showed that XB and OH-XB are promising new lead compounds for cancer therapy with distinct cellular mechanisms. Both compounds are candidates for further pre-clinical and clinical investigations.