Integrated analysis of gene expression and methylation profiles of novel pancreatic cancer cell lines with highly metastatic activity

Pancreatic cancer is one of the most lethal human malignancies, partly because of its propensity for metastasis. However, highly metastatic human pancreatic cancer cell lines suitable for studies of metastasis are currently lacking. Here we established two highly metastatic human pancreatic cancer cell lines, MIA PaCa-2 In8 and Panc-1 In8, by Matrigel induction assay. The cell lines were further characterized both in vitro and in vivo. MIA PaCa-2 In8 and Panc-1 In8 cells demonstrated increased migration and invasion compared with their respective parental cells. Following injection into nude mice, MIA PaCa-2 In8 and Panc-1 In8 cells resulted in more pulmonary metastases compared with the parental cells. Furthermore, analyses of m RNA, long non-coding RNA, micro RNA, and methylation profiling revealed that these factors were aberrantly regulated in the highly metastatic cells,indicating that they probably affected metastasis. We thus established and characterized two highly metastatic human pancreatic cell lines that could be used as valuable tools for future investigations into the pathogenesis, metastasis, and potential treatment of human pancreatic cancer.     

miR‐200c suppresses stemness and increases cellular sensitivity to trastuzumab in HER2+ breast cancer

Resistance to trastuzumab remains a major obstacle in HER2‐overexpressing breast cancer treatment. miR‐200c is important for many functions in cancer stem cells (CSCs), including tumour recurrence, metastasis and resistance. We hypothesized that miR‐200c contributes to trastuzumab resistance and stemness maintenance in HER2‐overexpressing breast cancer. In this study, we used HER2‐positive SKBR3, HER2‐negative MCF‐7, and their CD44⁺CD24^? phenotype mammospheres SKBR3‐S and MCF‐7‐S to verify. Our results demonstrated that miR‐200c was weakly expressed in breast cancer cell lines and cell line stem cells. Overexpression of miR‐200c resulted in a significant reduction in the number of tumour spheres formed and the population of CD44⁺CD24^? phenotype mammospheres in SKBR3‐S. Combining miR‐200c with trastuzumab can significantly reduce proliferation and increase apoptosis of SKBR3 and SKBR3‐S. Overexpression of miR‐200c also eliminated its downstream target genes. These genes were highly expressed and positively related in breast cancer patients. Overexpression of miR‐200c also improved the malignant progression of SKBR3‐S and SKBR3 in vivo. miR‐200c plays an important role in the maintenance of the CSC‐like phenotype and increases drug sensitivity to trastuzumab in HER2+ cells and stem cells.     

Doxorubicin‐induced cardiotoxicity is maturation dependent due to the shift from topoisomerase IIα to IIβ in human stem cell derived cardiomyocytes

Doxorubicin (DOX) is widely used to treat various cancers affecting adults and children; however, its clinical application is limited by its cardiotoxicity. Previous studies have shown that children are more susceptible to the cardiotoxic effects of DOX than adults, which may be related to different maturity levels of cardiomyocyte, but the underlying mechanisms are not fully understood. Moreover, researchers investigating DOX‐induced cardiotoxicity caused by human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) have shown that dexrazoxane, the recognized cardioprotective drug for treating DOX‐induced cardiotoxicity, does not alleviate the toxicity of DOX on hiPSC‐CMs cultured for 30 days. We have suggested that this may be ascribed to the immaturity of the 30 days hiPSC‐CMs. In this study, we investigated the mechanisms of DOX induced cardiotoxicity in cardiomyocytes of different maturity. We selected 30‐day‐old and 60‐day‐old hiPSC‐CMs (day 30 and day 60 groups), which we term ‘immature’ and ‘relatively mature’ hiPSC‐CMs, respectively. The day 30 CMs were found to be more susceptible to DOX than the day 60 CMs. DOX leads to more ROS (reactive oxygen species) production in the day 60 CMs than in the relatively immature group due to increased mitochondria number. Moreover, the day 60 CMs mainly expressed topoisomerase IIβ presented less severe DNA damage, whereas the day 30 CMs dominantly expressed topoisomerase IIα exhibited much more severe DNA damage. These results suggest that immature cardiomyocytes are more sensitive to DOX as a result of a higher concentration of topoisomerase IIα, which leads to more DNA damage.     

5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.     

Inhibition of circular RNA CDR1as increases chemosensitivity of 5‐FU‐resistant BC cells through up‐regulating miR‐7

This study aims to explore the mechanism of Circular RNA CDR1as implicating in regulating 5‐fluorouracil (5‐FU) chemosensitivity in breast cancer (BC) by competitively inhibiting miR‐7 to regulate CCNE1. Expressions of CDR1as and miR‐7 in 5‐FU‐resistant BC cells were determined by RT‐PCR. CCK‐8, colony formation assay and flow cytometry were applied to measure half maximal inhibitory concentration (IC50), 5‐Fu chemosensitivity and cell apoptosis. Western blot was used to detect the expressions of apoptosis‐related factors. CDR1as was elevated while miR‐7 was inhibited in 5‐FU‐resistant BC cells. Cells transfected with si‐CDR1as or miR‐7 mimic had decreased IC50 and colony formation rate, increased expressions of Bax/Bcl2 and cleaved‐Caspase‐3/Caspase‐3, indicating inhibition of CDR1as and overexpression of miR‐7 enhances the chemosensitity of 5‐FU‐resistant BC cells. Targetscan software indicates a binding site of CDR1as and miR‐7 and that CCNE1 is a target gene of miR‐7. miR‐7 can gather CDR1as in BC cells and can inhibit CCNE1. In comparison to si‐CDR1as group, CCNE1 was increased and chemosensitivity to 5‐Fu was suppressed in si‐CDR1as + miR‐7 inhibitor group. When compared with miR‐7 mimic group, CDR1as + miR‐7 mimic group had increased CCNE1 and decreased chemosensitivity to 5‐Fu. Nude mouse model of BC demonstrated that the growth of xenotransplanted tumour in si‐CDR1as + miR‐7 inhibitor group was faster than that in si‐CDR1as group. The tumour growth in CDR1as + miR‐7 mimic group was faster than that in miR‐7 mimic group. CDR1as may regulate chemosensitivity of 5‐FU‐resistant BC cells by inhibiting miR‐7 to regulate CCNE1.     

Deletion of pancreatic β‐cell adenosine kinase improves glucose homeostasis in young mice and ameliorates streptozotocin‐induced hyperglycaemia

Severe reduction in the β‐cell number (collectively known as the β‐cell mass) contributes to the development of both type 1 and type 2 diabetes. Recent pharmacological studies have suggested that increased pancreatic β‐cell proliferation could be due to specific inhibition of adenosine kinase (ADK). However, genetic evidence for the function of pancreatic β‐cell ADK under physiological conditions or in a pathological context is still lacking. In this study, we crossed mice carrying LoxP‐flanked Adk gene with Ins2‐Cre mice to acquire pancreatic β ‐cell ADK deficiency (Ins2‐Cre^±Adk^fl/fl) mice. Our results revealed that Ins2‐Cre^+/‐Adk^fl/fl mice showed improved glucose metabolism and β‐cell mass in younger mice, but showed normal activity in adult mice. Moreover, Ins2‐Cre^±Adk^fl/fl mice were more resistant to streptozotocin (STZ) induced hyperglycaemia and pancreatic β‐cell damage in adult mice. In conclusion, we found that ADK negatively regulates β‐cell replication in young mice as well as under pathological conditions, such as STZ induced pancreatic β‐cell damage. Our study provided genetic evidence that specific inhibition of pancreatic β‐cell ADK has potential for anti‐diabetic therapy.