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Systemic toxicity and tumor cell resistance still limit the efficacy of chemotherapy in colorectal cancer. Therefore, alternative treatments are desperately needed. The thiazolide Nitazoxanide (NTZ) is an FDA-approved drug for the treatment of parasite-mediated infectious diarrhea with a favorable safety profile. Interestingly, NTZ and the thiazolide RM4819—its bromo-derivative lacking antibiotic activity—are also promising candidates for cancer treatment. Yet the exact anticancer mechanism(s) of these compounds still remains unclear. In this study, we systematically investigated RM4819 and NTZ in 2D and 3D colorectal cancer culture systems. Both compounds strongly inhibited proliferation of colon carcinoma cell lines by promoting G1 phase cell cycle arrest. Thiazolide-induced cell cycle arrest was independent of the p53/p21 axis, but was mediated by inhibition of protein translation via the mTOR/c-Myc/p27 pathway, likely caused by inhibition of mitochondrial respiration. While both thiazolides demonstrated mitochondrial uncoupling activity, only RM4819 inhibited the mitochondrial respiratory chain complex III. Interestingly, thiazolides also potently inhibited the growth of murine colonic tumoroids in a comparable manner with cisplatin, while in contrast to cisplatin thiazolides did not affect the growth of primary intestinal organoids. Thus, thiazolides appear to have a tumor-selective antiproliferative activity, which offers new perspectives in the treatment of colorectal cancer. published

Colorectal cancer (CRC) is a frequently occurring malignant disease with still low survival rates, highlighting the need for novel therapeutics. Merosesquiterpenes are secondary metabolites from marine sponges, which might be useful as antitumor agents. To address this issue, we made use of a compound library comprising 11 isolated merosesquiterpenes. The most cytotoxic compounds were smenospongine &gt, ilimaquinone ≈ dactylospontriol, as shown in different human CRC cell lines. Alkaline Comet assays and γH2AX immunofluorescence microscopy demonstrated DNA strand break formation in CRC cells. Western blot analysis revealed an activation of the DNA damage response with CHK1 phosphorylation, stabilization of p53 and p21, which occurred both in CRC cells with p53 knockout and in p53-mutated CRC cells. This resulted in cell cycle arrest followed by a strong increase in the subG1 population, indicative of apoptosis, and typical morphological alterations. In consistency, cell death measurements showed apoptosis following exposure to merosesquiterpenes. Gene expression studies and analysis of caspase cleavage revealed mitochondrial apoptosis via BAX, BIM, and caspase-9 as the main cell death pathway. Interestingly, the compounds were equally effective in p53-wild-type and p53-mutant CRC cells. Finally, the cytotoxic activity of the merosesquiterpenes was corroborated in intestinal tumor organoids, emphasizing their potential for CRC chemotherapy.

In eukaryotic cells, the transport of cargo from the endoplasmic reticulum (ER) to the Golgi apparatus is conducted by protein-coated membrane vesicles. The stepwise formation of the coat protein complex II (COPII) on ER membranes is initiated by the small GTPase Sar1. Following GDP/GTP exchange, which is catalyzed by the guanine nucleotide exchange factor (GEF) Sec12, Sar1 undergoes a conformational change upon which an amphipathic helix inserts into the proximal leaflet of the ER membrane. Membrane-bound Sar1p subsequently recruits the inner and outer coat subcomplexes Sec23/Sec24 and Sec13/Sec31 to complete the COPII coat. This process leads to marked changes in membrane curvature. We apply in vitro reconstitution studies with purified yeast proteins on giant unilamellar vesicles (GUVs) as an artificial membrane system to examine the COPII vesicle formation process. Using confocal microscopy for visualization, we observe the formation of rigid tubes protruding from the GUVs under conditions where GTP hydrolysis is prevented. Cryo-electron microscopy shows tubules that apparently fail to fission into separate vesicles. Moreover, we can distinguish individual stages in the formation of the COPII coat on the bilayer. Altering the lipid composition, we observe different membrane morphologies. Our in vitro investigation of the COPII complex shows that GTP hydrolysis is not essential for binding of the coat to the membrane but may have a role in vesicle fission.