The Covid-19 pandemic has emerged as the leading public health challenge of our time (20.sup.th century). While vaccinations have finally blunted the death rate, concern has remained about more ...virulent forms highlighting the need for alternative approaches. Epidemiological studies indicate that physical activity has been shown to decrease the risk of infection of some respiratory viruses. Part of the salutary effects of exercise is believed to be through the elaboration of cytokines by contracting skeletal muscles (termed myokines). The objective of this study was to investigate whether exercise-induced myokines would mitigate the SARS-CoV-2 infectivity of the bronchial epithelium through modulating the SARS-CoV-2 Covid-19 receptor (angiotensin-converting enzyme 2 -ACE2) its priming enzyme, transmembrane serine protease 2 (TMPRSS2). We utilized a cell culture model of exercise to generate myokines by differentiating C2C12 cells into myotubules and inducing them to contract via low-frequency electric pulse stimulation. Condition media was concentrated via centrifugation and applied to human immortalized human bronchial epithelium cell line (6HBE14o) along with conditioned media from unstimulated myotubules as controls. Following exposure to myokines, the 16HBE14o cells were harvested and subjected to quantitative RT-PCR and Enzyme-Linked Immunosorbent Assay (ELISA) for assessment of mRNA and protein levels of ACE2 and TMPRSS2, respectively. Pilot proteomic data was performed with isotope barcoding and mass spectroscopy. Quantitative Real-Time PCR of 16HBE14o with 48 h treated unstimulated vs. stimulated myokine treatment revealed a reduction of ACE2 and TMPRSS2 mRNA by 32% (p<2.69x10.sup.-5) and 41% (p<4.57x10.sup.-5 ), respectively. The high sensitivity of ELISAs showed downregulation of ACE2 and TMPRSS2 protein expression in 16HBE14o cells by 53% (p<0.01) and 32% (p<0.03) respectively with 48 h treated. For rigor, this work was replicated in the human lung cancer cell line A549, which mirrored the downregulation. Proteomic analysis showed dramatic alteration in myokine profile between contracted and uncontracted C2C12 tubules. The current study explores a novel approach of a modified exercise cell culture system and uses ACE2 and TMPRSS2 as a surrogate marker of SARS-CoV-2 infectivity. In conclusion, we demonstrated biological data supporting exercise's protective effect against Covid-19. These further strengthen myokines' beneficial role as potential therapeutic targets against SARS-CoV-2 and similar viruses albeit these preliminary cell culture studies will require future validation in animal models.
Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin ...architecture and genome function. Specifically, the histone deacetylase (HDAC) family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC). However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs) interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA) targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS) to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.
IntroductionColorectal cancer (CRC) ranks as the second leading cause of cancer deaths in the UK. It is eminently preventable through identification and colonoscopic removal of the precursor lesion, ...the advanced adenoma (AAs). However, screening is both inefficient and inaccurate. For example, fecal immunohistochemical test (FIT) has a ~25% sensitivity for AAs. On the other hand, colonoscopy has a prevalence of ~6–8% meaning that >90% of screening colonoscopies are squandered with CRC prevention perspective. Finding better risk stratification is critical. Our group has focused on detection of field carcinogenesis, the notion that the genetic/epigenetic milieu that leads to a focal neoplasm can be detected throughout the colon including the readily accessible rectum. This is the clinical rationale for full colonoscopy for polyps on flex sigmoidoscopy or post-polypectomy surveillance (‘condemned mucosa’). MicroRNAs (miRs) are small (18–25 nucleotide) molecules that can impact specific gene expression. There are ~3000 species and miRs are resistant to degradation so ideal for detection in the colon.MethodsHuman Subjects: After IRB approved informed consent, we recruited patients undergoing colonoscopy and obtained 6 biopsies of the endoscopically normal rectal mucosa. Of the ~150 subjects we identified 9 with advanced adenomas elsewhere in the colon (cases) and selected 15 matched (negative colonoscopies) controls.RNA Seq: RNeasy mini kits (Ambion) for mRNA sequencing. Sequencing was performed using the Illumina® NovaSeqTM platform. Differential miR was analyzed. We assessed performance via the area under the receiver operator characteristic curve (AUROC).ResultsWe identified 2268 miRNA. Of those 165 miRNA values were statistically significant (p<0.05), were >1.2 fold or <0,8 dysregulated. There were 46 microRNAs downregulated and 119 upregulated. The top three miRs were hsa-miR-181b-5p with hsa-let-7d-3p and hsa-miR-4661–5p. The performance is in the table 1 below:Abstract O52 Table 1 AUROC miR-181b-5p0.81miR-181b-5p + hsa-let-7d-3p0.92miR-181b-5p+ hsa-let-7d-3p+ hsa-miR-4661–5p0.92We did leave one out validation to determine 95% confidence interval. For the single hsa-miR-181b-5p and with addition of hsa-let-7d-3p it was 0.76, 0.86 and 0.88, 0.95 respectively.ConclusionsWe show for the first time that a panel of 2 rectal miRs had an excellent performance for diagnosis. It is striking that this simple panel outperformed classic tests such as FIT or Cologuard (~25% and ~45% respectively). This proof of concept study suggests a simple rectal swab may be able to personalize CRC screening.
Hepatocellular carcinoma (HCC) results from chronic inflammation/cirrhosis. Unfortunately, despite use of radiological/serological screening techniques, HCC ranks as a leading cause of cancer deaths. ...Our group has used alterations in high order chromatin as a marker for field carcinogenesis and hence risk for a variety of cancers (including colon, lung, prostate, ovarian, esophageal). In this study we wanted to address whether these chromatin alterations occur in HCC and if it could be used for risk stratification.
A case control study was performed in patients with cirrhosis who went on to develop HCC and patients with cirrhosis who did not develop cancer. We performed partial wave spectroscopic microscopy (PWS) which measures nanoscale alterations on formalin fixed deparaffinized liver biopsy specimens, 17 progressors and 26 non-progressors. Follow up was 2089 and 2892 days, respectively.
PWS parameter disorder strength Ld were notably higher for the progressors (Ld = 1.47 ± 0.76) than the non-progressors (Ld = 1.00 ± 0.27) (p = 0.024). Overall, the Cohen's d effect size was 0.907 (90.7%). AUROC analysis yielded an area of 0.70. There was no evidence of confounding by gender, age, BMI, smoking status and race.
High order chromatin alterations, as detected by PWS, is altered in pre-malignant hepatocytes with cirrhosis and may predict future risk of HCC.
Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. There is an increasing need for the identification of biomarkers of pre-malignant and early stage ...CRC to improve risk-stratification and screening recommendations. In this study, we investigated the possibility of metabolic and mitochondrial reprogramming early in the pre-malignant colorectal field.
Rectal biopsies were taken from 81 patients undergoing screening colonoscopy, and gene expression of metabolic and mitochondrial markers were assessed using real time quantitative PCR. Validation studies were performed in two different animal models of colon carcinogenesis: Pirc rats and AOM-treated rats.
We found evidence of a Warburg effect in the normal-appearing rectal mucosa of patients harboring precancerous lesions elsewhere in the colon compared to control patients, with a significant increase in HIF1α, SLC2A1 (referred to as GLUT1), PKM2, and LDHA. We also found evidence of early mitochondrial changes in the colorectal field of patients harboring pre-cancerous lesions, with significantly increased mitochondrial gene expression of DRP1 (fission), OPA1 (fusion), PGC1-α (biogenesis), UCP2 (uncoupling) and mtND1 (copy number). Similar results were observed in the two different animal models.
These results demonstrate for the first time evidence of early Warburg-like metabolic changes as well as changes in mitochondrial function, dynamics and mtDNA copy number in endoscopically normal premalignant colorectal mucosal field. These findings provide an opportunity for the development of metabolic biomarkers that could be used for improving screening recommendations and risk-stratification. This also provides a potential target for novel chemopreventive strategies in the pre-malignant colorectal field.
•EB1 expression is significantly increased in early and field carcinogenesis in the colon.•Knockdown of EB1 reduced the cancerous phenotype in colon cancer cell lines.•LEBS analysis of EB1 knockdown ...cells shows distinct changes in cell nano-architecture.
End-binding protein (EB1) is a microtubule protein that binds to the tumor suppressor adenomatous polyposis coli (APC). While EB1 is implicated as a potential oncogene, its role in cancer progression is unknown. Therefore, we analyzed EB1/APC expression at the earliest stages of colorectal carcinogenesis and in the uninvolved mucosa (“field effect”) of human and animal tissue. We also performed siRNA-knockdown in colon cancer cell lines. EB1 is up-regulated in early and field carcinogenesis in the colon, and the cellular/nano-architectural effect of EB1 knockdown depended on the genetic context. Thus, dysregulation of EB1 is an important early event in colon carcinogenesis.
Nuclear alterations are a well-known manifestation of cancer. However, little is known about the early, microscopically-undetectable stages of malignant transformation. Based on the phenomenon of ...field cancerization, the tissue in the field of a tumor can be used to identify and study the initiating events of carcinogenesis. Morphological changes in nuclear organization have been implicated in the field of colorectal cancer (CRC), and we hypothesize that characterization of chromatin alterations in the early stages of CRC will provide insight into cancer progression, as well as serve as a biomarker for early detection, risk stratification and prevention.
For this study we used transmission electron microscopy (TEM) images of nuclei harboring pre-neoplastic CRC alterations in two models: a carcinogen-treated animal model of early CRC, and microscopically normal-appearing tissue in the field of human CRC. We quantify the chromatin arrangement using approaches with two levels of complexity: 1) binary, where chromatin is separated into areas of dense heterochromatin and loose euchromatin, and 2) grey-scale, where the statistics of continuous mass-density distribution within the nucleus is quantified by its spatial correlation function.
We established an increase in heterochromatin content and clump size, as well as a loss of its characteristic peripheral positioning in microscopically normal pre-neoplastic cell nuclei. Additionally, the analysis of chromatin density showed that its spatial distribution is altered from a fractal to a stretched exponential.
We characterize quantitatively and qualitatively the nanoscale structural alterations preceding cancer development, which may allow for the establishment of promising new biomarkers for cancer risk stratification and diagnosis. The findings of this study confirm that ultrastructural changes of chromatin in field carcinogenesis represent early neoplastic events leading to the development of well-documented, microscopically detectable hallmarks of cancer.
PDF
