Knock-Down of Core Proteins Regulating MicroRNA Biogenesis Has No Effect on Sensitivity of Lung Cancer Cells to Ionizing Radiation

Recent studies underline the important role of microRNAs (miRNA) in the development of lung cancer. The main regulators of miRNA biogenesis are the ribonucleases Drosha, Dicer and Ago2. Here the role of core proteins of miRNA biogenesis machinery in the response of human non-small and small cell lung carcinoma cell lines to treatment with ionizing radiation was assessed. We found that Drosha and Dicer were expressed at higher levels in radioresistant but not in sensitive cell lines. However, down-regulation of either Dicer or Drosha had no effect on the sensitivity of cells to irradiation. Elimination of components of the RNA-induced silencing complex Ago2 and Tudor staphylococcal nuclease also did not sensitize cells to the same treatment. Thus, modulation of miRNA biogenesis machinery is not sufficient to increase the radiosensitivity of lung tumors and other strategies are required to combat lung cancer

Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer

Background: Hyaluronan (HA) an important component of the extracellular matrix, has been linked to tumor progression and drug resistance in several malignancies. However, limited data is available for ovarian cancer. This study investigated the role of hyaluronan (HA) and a potential link between the HA-CD44 pathway and membrane ATP binding cassette (ABC) transporter proteins in ovarian cancer chemoresistance. Methods: We investigated the ability of HA to block the cytotoxic effects of the chemotherapy drug carboplatin, and to regulate the expression of ABC transporters in ovarian cancer cells. We also examined HA serum levels in ovarian cancer patients prior to and following chemotherapy and assessed its prognostic relevance. Results: HA increased the survival of carboplatin treated ovarian cancer cells expressing the HA receptor, CD44 (OVCAR-5 and OV-90). Carboplatin significantly increased expression of HAS2, HAS3 and ABCC2 and HA secretion in ovarian cancer cell conditioned media. Serum HA levels were significantly increased in patients following platinum based chemotherapy and at both 1st and 2nd recurrence when compared with HA levels prior to treatment. High serum HA levels (>50 μg/ml) prior to chemotherapy treatment were associated with significantly reduced progression-free (P = 0.014) and overall survival (P = 0.036). HA production in ovarian cancer cells was increased in cancer tissues collected following chemotherapy treatment and at recurrence. Furthermore HA treatment significantly increased the expression of ABC drug transporters (ABCB3, ABCC1, ABCC2, and ABCC3), but only in ovarian cancer cells expressing CD44. The effects of HA and carboplatin on ABC transporter expression in ovarian cancer cells could be abrogated by HA oligomer treatment. Importantly, HA oligomers increased the sensitivity of chemoresistant SKOV3 cells to carboplatin. Conclusions: Our findings indicate that carboplatin chemotherapy induces HA production which can contribute to chemoresistance by regulating ABC transporter expression. The HA-CD44 signaling pathway is therefore a promising target in platinum resistant ovarian cancer.Carmela Ricciardelli, Miranda P Ween, Noor A Lokman, Izza A Tan, Carmen E Pyragius, and Martin K Oehle

Topoisomerase II alpha, Multidrug Resistance Associated Protein-1 und Dicer- Expression als prognostische Faktoren im Ovarialkarzinom

Integration of biomarker analysis in tumor tissue with conventional clinical and pathological diagnostics could provide a better insight into the prognosis of ovarian cancer patients. In this research project, a new technique for RNA isolation from formalin-fixed paraffin- embedded tissue (FFPE) followed by real time RT-PCR was used to quantitatively assess potential prognostic biomarkers in ovarian cancer. Furthermore, biomarker expression was evaluated at the protein level by immunohistochemistry. The biomarkers studied included: topoisomerase II alpha (Top IIα), multidrug resistance-associated protein 1 (MRP1), insulin-like growth factor-II mRNA binding protein 3 (IMP3), estrogen receptor alpha (ERα) and Dicer. Biomarker expression was associated with patient survival and with clinicopathological variables. A positive correlation between expression of ERα, Dicer and IMP3 with overall survival times was observed, whereas Top IIα and MRP1 expression was linked to an unfavourable prognosis. The findings indicate the prognostic value of these biomarkers in ovarian cancer and suggest their contribution to molecular events underlying ovarian cancer. Furthermore, we provide data on the validation of the assessment of biomarkers at the RNA level in FFPE tissue. Molecular biomarker evaluation may help assess patients’ risk profile and planning individually-tailored therapy.Die Verbindung von Biomarkeranalyse im Tumorgewebe mit konventioneller klinischer und pathologischer Diagnostik verspricht eine genauere Prognoseabschätzung bei Ovarialkarzinompatientinnen. Im hier vorgestellten Forschnugsprojekt wurde eine neue Technik zur RNA-Isolation aus Formalin- fixiertem, Paraffin-eingebetteten (FFPE) Gewebe und Analyse mittels real time RT-PCR verwendet, um quantitativ potentielle prognostische Marker im Ovarialkarzinom zu bestimmen. Außerdem wurde die Proteinexpression dieser Marker mittels Immunhistochemie untersucht. Die untersuchten Biomarker waren: Topoisomerase II alpha (Top IIα), Multidrug resistance-associated protein 1 (MRP1), Insulin-like growth factor-II mRNA binding protein 3 (IMP3), Östrogenrezeptor alpha (ERα) und Dicer. Die Expression der genannten Biomarker war mit der Überlebenszeit und verschiedenen klinisch-pathologischen Parametern assoziiert. Hierbei wurde eine positive Assoziation zwischen der Expression von ERα, Dicer und IMP3 und der Prognose gefunden, wohingegen die Expression von Top IIα und MRP1 mit einer schlechten Prognose verbunden war. Diese Ergebnisse zeigen den prognostischen Wert dieser molekularen Marker beim Ovarialkarzinom und weisen darauf hin, daß die betreffenden Moleküle an der Tumorentstehung und -progression beteiligt sein könnten. Zudem wurde einen neue Methode der Biomarkeranalyse auf RNA Ebene im FFPE Gewebe validiert. Die Expressionsanalyse dieser Biomarker könnte dazu beitragen, die Patientenprognose besser abzuschätzen und damit eine individuelle Therapie zu planen

C-reactive protein is associated with low-density lipoprotein cholesterol and obesity in type 2 diabetic Sudanese

Angelo C Dongway,1 Areeg S Faggad,2,3 Hani Y Zaki,2 Badreldin E Abdalla,2,41Department of Biochemistry, Faculty of Medicine, Upper Nile University, Malakal, South Sudan; 2Department of Biochemistry and Nutrition, Faculty of Medicine, University of Gezira, 3Department of Molecular Biology, National Cancer Institute-University of Gezira, Wad Medani, Sudan; 4Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia Background: Type 2 diabetes is emerging in Sudan and is associated with obesity. Deregulated lipid metabolism and inflammatory states are suggested risk factors for cardiovascular disease, which is a leading cause of diabetic death. This study aimed to investigate C-reactive protein (CRP) levels and the lipid profile in type 2 diabetic adult Sudanese compared with nondiabetics, and to test their associations with other characteristics. Methods: A cross-sectional study including 70 diabetics and 40 nondiabetics was conducted. Anthropometric measurements were assessed, and demographic and medical data were obtained using a structured questionnaire. Blood specimens were collected and biochemical parameters were analyzed applying standard methods. Results: CRP and triglycerides were significantly higher in the diabetic group (P<0.001 and P=0.01, respectively). Differences in total cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were not statistically significant between the diabetic and nondiabetic groups. In the diabetic group, correlation analysis revealed that the CRP level had a significant positive correlation with LDL-C (r=0.255, P=0.034) and body mass index (r=0.29, P=0.016). Body mass index showed a significant positive correlation with triglycerides (r=0.386, P=0.001). Within the lipid parameters, a number of significant correlations were observed. Elevated levels of CRP, LDL-C, and triglycerides were markedly more prevalent in the diabetic group of patients. Diabetics showed significantly higher CRP levels compared with nondiabetics (odds ratio 5.56, P=0.001). Conclusion: The high prevalence of obesity among diabetics, together with elevated levels of triglycerides and CRP, suggest coexistence of dyslipidemia and inflammation in diabetes. Our findings emphasize that diabetics were 5.6 times more likely to have high CRP levels than nondiabetics; as CRP is a predictor of cardiovascular disease risk, it can be recognized that diabetics are at more risk of cardiovascular disease than nondiabetics. Considering evaluation of CRP together with the lipid profile in prediction of cardiovascular disease risk in Sudanese diabetics should be further tested in large-scale studies. Keywords: C-reactive protein, type 2 diabetes, lipid profile, obesity, cardiovascular disease, Sudanes

Combination of Nanovectorized siRNA Directed against Survivin with Doxorubicin for Efficient Anti-Cancer Activity in HER2+ Breast Cancer Cells

International audienceAccording to Globocan 2020, breast cancer is considered one of the most common cancers affecting women and is one of the leading causes of death in over 100 countries. The available classical treatment options do not always give satisfactory outcomes, and some patients develop resistance to these treatments. This study aims to investigate the combination of nanovectorized siRNA directed against anti-apoptotic protein Survivin (siSurvivin) by targeted stealth magnetic siRNA nanovectors (TS-MSN), designed in our lab, with Doxorubicin (DOX), as an option for HER2+ breast cancer treatment. The hypothesis is that the pretreatment of the HER2+ breast cancer cell line SK-BR-3 with siSurvivin will induce apoptosis in the cancer cells and enhance the therapeutic efficacy of DOX, allowing a dose reduction of DOX and hence a reduction of potential side effects. TS-MSN are based on superparamagnetic iron oxide nanoparticles (SPIONs) covalently coupled with a fluorophore sulfocyanine-5 and polyethylene glycol 5000 (PEG5000) and functionalized with single-chain variable fragments (scFv) of an antibody targeting the HER2 membrane receptor. These covalently functionalized SPIONs are then complexed via electrostatic interactions with therapeutic siRNA and the cationic polymers, chitosan, and poly-L-arginine. TS-MSNsiSurvivin had an average size of 144 ± 30 nm, a PDI of 0.3, and a slightly positive zeta potential value of 10.56 ± 05.70 mV. The agarose gel electrophoresis assay confirmed that the siRNA is well-complexed into TS-MSN without leakage, as no free siRNA was detected. Moreover, siRNA in TS-MSN was protected from RNAse A degradation for up to 6 h at 37 °C. Formulations of TS-MSN with siSurvivin demonstrated in vitro gene knockdown up to 89% in the HER2+ breast cancer cell line SK-BR-3. Furthermore, qRT-PCR confirmed a significant Survivin mRNA relative expression inhibition (about 50%) compared to control siRNA or untreated cells. A combination protocol was evaluated between TS-MSN and Doxorubicin (DOX) for the first time. Therefore, SK-BR-3 cells were pretreated with TS-MSN formulated with siSurvivin at 50 nM for 24 h alone, before a DOX treatment at a concentration of 0.5 µM (corresponding to the IC50) was added for 48 h. The MTT cytotoxicity tests, performed after 72 h of treatment, revealed that the combination had a significant synergistic cytotoxic effect on SK-BR-3 cells compared to monotherapies or untreated cells. We confirmed that pretreatment of cells with siSurvivin potentializes the cytotoxic effect of DOX as an alternative approach for treating HER2+ breast cancer. In conclusion, a combination of anti-Survivin siRNA and DOX would be a good alternative in HER2+ breast cancer therapy