The Effect of Disrupted Circadian Rhythm and Associated microRNA on Biliary Injury and Malignant Transformation

Cholangiocarcinoma (CCA) is a devastating tumor characterized by late presentation of symptoms with limited treatment options. Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small noncoding RNAs that trigger mRNA translation, repression or degradation. The aim of the study was to evaluate the role of deregulated circadian rhythm and related microRNAs in CCA growth. Human intra- and extrahepatic CCA cells and non-malignant (H69) human cholangiocytes were serum starved for 48 hours before stimulation with 50% serum for 2 hours. The 24-hours rhythmic expression of core clock genes, such as Per1/2/3, CLOCK, Bmal1, Cry1/2 and two clock-controlled genes (CCGs) WEE1 and DBP, was evaluated in the selected CCA cells and H69 controls by real-time PCR. To further evaluate the role of Per1, we overexpressed Per1 by transfecting Mz-ChA-1 CCA cells with Per1 or empty vector. In parallel studies, we silenced miR-34a expression with anti-miR-34a inhibitor. Then, we measured: (i) cell proliferation by MTS assays and PCNA immunoblots; (ii) cell cycle; (iii) apoptosis; and (iv) cell migration and. We used luciferase assay to demonstrate whether Per1 acts as a direct target of miR-34a. Finally, we maintained CCA xenograft nude mice in complete dark or light/dark cycle for up to 40 days before evaluating tumor growth. We found the 24-hours rhythmical expression of Per1 was abolished in all CCA cell lines. The rhythmic expression of Bmal1, CLOCK, Per2/3, Cry1/2, WEE1 and DBP was also lost in some of the CCA cell lines tested. After overexpression of Per1, Mz-ChA-1 showed: (i) reduced cell proliferation; (ii) higher G0/G1 arrest and lower G2/M arrest and (iii) enhanced apoptosis. miR-34a was rhythmically expressed in CCA cell lines and H69. Moreover, the inhibition of miR-34a decreased proliferation, migration and invasion in the selected CCA cell lines. Per1 was verified as a target of miR-34a. However, prolonged darkness therapy did not inhibit the CCA xenograft growth in vivo. Summary and conclusions: Disruption of circadian rhythms contributes to the malignant phenotypes of human CCA, and may serve as novel prognostic or therapeutic targets for CCA

The Role of Creatine Supplementation in Alleviating Doxorubicin Induced Hepatotoxicity

Doxorubicin AKA “The red devil” is a commonly used chemotherapeutic drug. It induces significant oxidative injury at the cellular level and is routinely used for aggressive forms of cancer. Severe side effects of doxorubicin include cardiac and liver damage. Mitigating the severity of these side effects is an important part of ongoing research. It has the potential to improve patients’ lives and long-term life expectancy. Creatine is a considered a safe ergogenic substance and recently has been suggested as a potential therapeutic for doxorubicin induced side effects. To explore this potential benefit, Sprague-Dawley rats were used as a model organism. Six treatment groups consisting of four rats each received either saline, doxorubicin, 2% creatine/saline, 2%creatine/doxorubicin, 4/2%creatine/saline, 4/2% creatine/doxorubicin. The treatment groups were anesthetized and sacrificed after treatment. Liver samples were then collected. Tissue was snap frozen with liquid nitrogen. Liver function was examined by serum chemistry. Liver to body-weight ratio was calculated. H&E staining and Sirius red staining were used to examine the liver damage and liver fibrosis histologically. Genomic DNA and total RNA was also isolated and used to examine global methylation and apoptotic, liver fibrosis, oxidative, and inflammation biomarkers through ELISA and qPCR. The liver bodyweight ratio with the doxorubicin treatment presented significantly higher. 4/2% creatine/doxorubicin treatment recovered, while the 2% creatine/doxorubicin treatment significantly atrophied. Apoptosis, proliferation, and fibrosis increased in the 2% creatine/doxorubicin treatment while no significant increases occurred in the 4/2% creatine/doxorubicin treatments. Further, hypomethylation was shown in the doxorubicin treatment, while the 2% and 4/2% creatine/doxorubicin helped increase methylation levels. Together these results indicate a dose dependent effect of creatine supplementation with lower doses driving increased levels of stress and high doses improving liver phenotype. Our results indicate that overall liver health is improved as a result of creatine supplementation

Circadian rhythm and melatonin in liver carcinogenesis: updates on current findings

: Liver cancer, including hepatocellular carcinoma and cholangiocarcinoma, can be devastating if not treated early. The risk factors of liver cancer include alcoholic liver disease, non-alcoholic fatty liver disease, disruption of melatonin levels, and dysregulated circadian rhythm. The circadian rhythm is a 24-hour biological clock that regulates the physiological activities at both central and peripheral levels. Its molecular mechanism exists in every cell in mammals. Disruption of the circadian rhythm has found in liver cancers as an independent risk factor. This review summarized the most recent findings about the molecular mechanisms of circadian rhythm, the crosstalk between core clock genes and melatonin, as well as the role of circadian rhythm and melatonin played in chronic liver diseases and liver cancer. Finally, we discussed the potential clinical application of circadian rhythm and melatonin for the treatment of liver cancer and discussed future perspectives of how understanding the circadian rhythm in liver cancer progression could provide new clinical applications for liver cancer treatment and diagnosis