Mutational analysis of the enzymatic domain of Clostridium difficile toxin B reveals novel inhibitors of the wild-type toxin

Toxin B (TcdB), a major Clostridium difficile virulence factor, glucosylates and inactivates the small GTP-binding proteins Rho, Rac, and Cdc42. In the present study we provide evidence that enzymatically inactive fragments of the TcdB enzymatic domain are effective intracellular inhibitors of native TcdB. Site-directed and deletion mutants of the TcdB enzymatic region (residues 1 to 556), lacking receptor binding and cell entry domains, were analyzed for attenuation of glucosyltransferase and glucosylhydrolase activity. Five of six derivatives from TcdB(1-556) were found to be devoid of enzymatic activity. In order to facilitate cell entry, mutants were genetically fused to lfn, which encodes the protective antigen binding region of anthrax toxin lethal factor and mediates the cell entry of heterologous proteins. In line with reduced enzymatic activity, the mutants also lacked cytotoxicity. Remarkably, pretreatment or cotreatment of cells with four of the mutants provided protection against the cytotoxic effects of native TcdB. Furthermore, a CHO cell line expressing enzymatically active TcdB(1-556) was also protected by the mutant-derived inhibitors, suggesting that inhibition occurred at an intracellular location. Protection also was afforded by the inhibitor to cells treated with Clostridium sordellii lethal toxin (TcsL), which uses the same cosubstrate as TcdB but shares Rac only as a common substrate target. Finally, the inhibitor did not provide protection against Clostridium novyi alpha-toxin (Tcnalpha), which shares similar substrates with TcdB yet uses a different cosubstrate. This is the first report to demonstrate that the potential exists to inhibit toxins at their intracellular site of action by using inactive mutants.     

Higher magnification lenses versus conventional lenses for evaluation of diabetic neuropathy by corneal in vivo confocal microscopy

We investigated the association of CDKAL1 (rs7754840 and rs7756992) and CDKN2A/2B (rs10811661) variants with T2DM. Higher MAF of rs7754840 and rs7756992 were seen in patients, and both were associated with T2DM under additive, dominant, and recessive models. CDKAL1 rs7754840 and rs7756992, but not CDKN2A/2B rs10811661, are associated with T2DM in Lebanese.     

Evaluation of the bioequivalence and food effect on the bioavailability of CC-486 (oral azacitidine) tablets in adult patients with cancer

Cancer Chemotherapy and Pharmacology - CC-486 is an oral formulation of azacitidine that allows for extended dosing schedules to prolong azacitidine exposure to malignant cells and maximize...     

Chemotherapy rechallenge in metastatic colon cancer: A case report and literature review

BACKGROUNDColorectal cancer (CRC) is the third leading cause of cancer-related death in males and females in the United States. Approximately, 20%-22% of patients have metastatic disease at the time of presentation, and 50%-60% will develop metastasis over the course of their disease. Despite advances in systemic therapies, there remains a paucity of effective third- and later-line therapies for patients with ongoing disease progression. However, rechallenging chemo-resistant CRC tumors with previously administered therapies is an emerging concept that may be a life-prolonging option for heavily treated metastatic colorectal cancer (mCRC).CASE SUMMARYA 41-year-old man with no previous medical history initially presented with worsening diffuse abdominal tenderness. Computed tomography was significant for a splenic flexure mass and hepatic lesions concerning for metastatic disease. He underwent a colectomy with anastomosis. Postoperative pathology was diagnostic for moderately to well-differentiated adenocarcinoma (T4bN1bM1a). He received adjuvant 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX), but therapy was discontinued due to the development of atrial fibrillation. Additional workup indicated a carcinoembryonic antigen level of 508.2 ng/mL, and mutational analysis found that the tumor was microsatellite instability-high and KRAS/BRAF wild-type. He was started on irinotecan with oxaliplatin (IROX), and bevacizumab (14 cycles), developed disease progression, was transitioned to FOLFOX and cetuximab, and then eventually three cycles of pembrolizumab. Following disease progression, he was rechallenged with IROX therapy, as he previously responded well to oxaliplatin-based therapy. The IROX rechallenge provided this patient with a ten-month survival benefit, decreased metastatic burden, and marked improvement in his clinical condition.CONCLUSIONRechallenge of previous lines of well-tolerated systemic chemotherapy regimens may be a valuable therapeutic strategy in patients with heavily-treated mCRC.     

Giant multilocular prostatic cystadenoma in a young man: A case report from Jordan

Giant Multilocular Prostatic Cystadenoma (GMPC) is one of the rare benign tumors of the prostate. This report presents a case of a young man who has been recently diagnosed with GMPC. Our report highlights the importance of timely diagnosis and treatment, considering the overlapping symptoms with other common urinary conditions.     

Cyclooxygenase-2 inhibitors decrease vascular endothelial growth factor expression in colon cancer cells by enhanced degradation of Sp1 and Sp4 proteins

Cyclooxygenase 2 (COX-2) inhibitors, such as celecoxib (Cel), nimesulfide (NM), and NS-398 [NS; N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide] and other nonsteroidal anti-inflammatory drugs inhibit colon cancer growth and angiogenesis; however, the mechanism of this response is not well defined. Treatment of SW-480 colon cancer cells with Cel, NS, or NM decreased vascular endothelial growth factor (VEGF) mRNA and immunoreactive protein expression. This was also accompanied by decreased transactivation in cells transfected with constructs containing VEGF gene promoter inserts. Deletion analysis of the VEGF promoter indicated that decreased VEGF expression by COX-2 inhibitors was associated with the proximal -131 to -47 GC-rich region of the VEGF promoter that binds Sp proteins. Treatment of SW-480 cells with Cel, NM, and NS also decreased Sp1 and Sp4 protein expression but not that of Sp2 or Sp3. Similar results were observed in RKO, HT-29, and DLD colon cancer cells demonstrating comparable responses in COX-2-expressing and -nonexpressing colon cancer cell lines. COX-2 inhibitors do not affect Sp1 or Sp4 mRNA levels in SW-480 cells; however, decreased expression of both proteins was accompanied by increased protein ubiquitination and inhibited by the proteasome inhibitor gliotoxin. These results suggest that the antiangiogenic activity of COX-2 inhibitors in colon cancer cells is linked to activation of proteasome-dependent degradation of Sp1 and Sp4 proteins.     

5,5'-Dibromo-bis(3'-indolyl)methane induces Kruppel-like factor 4 and p21 in colon cancer cells

Bis(3'-indolyl)methane (DIM) is a metabolite of the phytochemical indole-3-carbinol, and both compounds exhibit a broad spectrum of anticancer activities. We have developed a series of synthetic symmetrical ring-substituted DIM analogues, including 5,5'-dibromoDIM, which are more potent than DIM as inhibitors of cancer cell and tumor growth. In colon cancer cells, 5,5'-dibromoDIM decreased cell proliferation and inhibited G(0)-G(1)- to S-phase progression, and this was accompanied by induction of the cyclin-dependent kinase inhibitor p21 in HT-29 and RKO colon cancer cells. Mechanistic studies showed that induction of p21 in both RKO (p53 wild-type) and HT-29 (p53 mutant) cells by 5,5'-dibromoDIM was Krüppel-like factor 4 (KLF4) dependent, and induction of p53 in RKO cells was also KLF4 dependent. Analysis of the p21 promoter in p53-dependent RKO cells showed that 5,5'-dibromoDIM activated p21 gene expression through the proximal GC-rich sites 1 and 2, and chromatin immunoprecipitation assays showed that KLF4 and p53 bound to this region of the promoter, whereas in HT-29 cells unidentified upstream cis-elements were required for induction of p21. 5,5'-DibromoDIM (30 mg/kg/d) also inhibited tumor growth and induced p21 in athymic nude mice bearing RKO cells as xenografts, showing that ring-substituted DIM such as 5,5'-dibromoDIM represent a novel class of mechanism-based drugs for clinical treatment of colon cancer.