Description:

The high host genetic background of tissue biopsies hinders the application of shotgun metagenomic sequencing in characterizing the tissue microbiota. We proposed an optimized method that removed host DNA from colon biopsies and examined the effect on metagenomic analysis. Human or mouse colon biopsies were divided into two groups, with one group undergoing host DNA depletion while the other serving as the control. Host DNAs were removed through differential lysis of mammalian and bacterial cells before sequencing. The impact of host DNA depletion on microbiota was compared based on phylogenetic diversity analyses and regression analyses. Removing host DNA enhanced bacterial sequencing depth and improved species discovery, increasing bacterial reads by 2.46 ± 0.20 fold while reducing host reads by 6.80% ± 1.06%. Moreover, 3.40 times more of bacterial species were detected after host DNA depletion. This was confirmed from mouse colon tissues, increasing bacterial reads by 5.46 ± 0.42 fold while decreasing host reads by 10.2% ± 0.83%. Similarly, significantly more species were detected in mouse colon tissue upon host DNA depletion (P < 0.001). Furthermore, an increased microbial richness was evident in the host DNA-depleted samples compared to non-depleted controls in human colon biopsies and mouse colon tissues (P < 0.001). Our optimized method of host DNA depletion improved the sensitivity of shotgun metagenomic sequencing in bacterial detection in the biopsy, which may yield a more accurate taxonomic profile of the tissue microbiota and identify bacteria that are important for disease initiation or progression. Colorectal cancer (CRC) is estimated as the third most incident cancer and second in mortality worldwide. Moreover, CRC metastasis reduces patients' survival rates. Thus, the study and identification of new compounds with anticancer activity selectively to tumor cells are encouraged in the CRC treatment. Naphtoquinones are compounds with several pharmacologic activities, including antitumoral properties. Therefore, this study aimed to investigate the anticancer mechanism of synthetic 8-Hydroxy-2-(P-Nitrothiophenol)-1, 4-Naphthoquinone (CNN16) in colon cancer cell line HCT-116. CNN16 showed an IC50 of 5.32 μM in HCT-116, and 9.36, 10.77, and 24.57 μM in the non-cancerous cells MRC-5, MNP-01, and PMBC, respectively, evaluated by the MTT assay. CNN16 showed an anticlonogenic effect in HCT-116 and induced cell fragmentation identified by flow cytometry analysis. Furthermore, we observed that CNN16 presented genotoxicity and induces Reactive Oxygen Species (ROS) after 3 h of treatment visualized by alkaline comet assay and DCFH-DA dye fluorescence, respectively. Furthermore, CNN16 caused cellular membrane disruption, reduction in the mitochondrial membrane polarization, and the presence of apoptotic bodies and chromatin condensation was visualized by differential stained (HO/FD/PI) in fluorescent microscopy along with PARP1, TP53, BCL-2, and BAX analyzed by RT-qPCR. Results also evidenced inhibition in the migratory process analyzed by wound healing assay. Therefore, CNN16 can be considered as a potential new leader molecule for CRC treatment, although further studies are still necessary to comprehend the effects of CNN16 in in vivo models to evaluate the anti-migratory effect, and toxicology and assure compound safety and selectively.

With Regards
Merry
Journal Coordinator
Global Journal of Research and Review