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

Doxorubicin and etoposide sensitize small cell lung carcinoma cells expressing caspase-8 to TRAIL

Abstract Background TRAIL is considered as a promising anti-cancer agent, because of its ability to induce apoptosis in cancer but not in most normal cells. However, growing evidence exist that many cancer cells are resistant to its apoptotic effects. SCLC is a typical example of tumor entity where TRAIL monotherapy is not efficient. Results We demonstrated that doxorubicin and etoposide markedly sensitized SCLC cells expressing caspase-8 to apoptotic effects of TRAIL. The drug-mediated sensitization of these cells was associated with increase of surface and total DR5 protein level, specific cleavage of cFLIPL, decrease of cFLIPS level, and a strong activation of caspase-8. The involvement of mitochondria-mediated pathway was demonstrated by enhanced Bid cleavage, Bax activation, and cytochrome c release. Activation of caspase-8 induced by combined treatment was shown to occur upstream of mitochondria and effector caspases. Conclusions Our results highlight significant applicability of doxorubicin and etoposide in sensitization of SCLC cells expressing caspase-8 to treatment with TRAIL.</p

The knock-down of a component of the RNA-induced silencing complex (RISC), Tudor-SN, is insufficient to sensitize NSCLC to irradiation.

<p>The level of Tudor-SN expression and cleavage of PARP in U1810 (A), A549 (C) and H661 (D) cells transfected (48 h) with control (si scr) or Tudor-SN (si TSN) siRNA analyzed by Western blot 48 h after irradiation. Equal loading was verified using anti-GAPDH antibodies. (B) The apoptotic cell death in U1810 cells after transfection with TSN siRNA and irradiation. All data are representative of three independent experiments.</p

Knock-down of Dicer and Drosha is not sufficient to sensitize NSCLC cells to irradiation.

<p>(A) The level of Dicer expression, cleavage of PARP and processing of caspase-3 and -9 in U1810 cells transfected (48 h) with control (si scr) or Dicer (siDicer) siRNA assessed by Western blot 48 h after irradiation. Equal loading was verified using anti-GAPDH antibodies. Data are representative of three independent experiments. (B) Detection of apoptotic cell death in U1810 assessed by measuring the sub-G1 population after transfection (48 h) with control or Dicer siRNA and irradiation treatment (48 h). (C) Caspase-3-like activity (fold increase with respect to control) in U1810 cells after treatment with either irradiation alone or in combination with transfection of control or Dicer siRNA (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033134#s2" target="_blank">Materials and methods</a>). (D) The level of Drosha expression and cleavage of PARP in U1810 cells transfected (48 h) with control (si scr) or Drosha (siDrosha) siRNA analyzed by Western blot 48 h after treatment with irradiation. Equal loading was verified using anti-GAPDH antibodies. All data are representative of three independent experiments. (B) Apoptotic cell death in U1810 measured by analysis of the sub-G1 population after transfection (48 h) with control or Drosha siRNA and irradiation treatment (48 h). Results shown are the mean±S.E.M. of three independent experiments.</p

NSCLC and SCLC cells differ in sensitivity to radiation treatment and display differential expression of proteins involved in the regulation of miRNA biogenesis.

<p>(A) Western blot analysis of the level of protein expression of Drosha, Dicer, exportin 5 (XPO5), Tudor-SN (TSN), protein activator of the interferon-induced protein kinase (PACT), fragile X mental retardation syndrome-related protein 1 (FXR1) and Argonaute 2 (AGO2) in a panel of NSCLC (U1810, U1299, A549, H661, H157, H23) and SCLC (U1285, H82, H69, U1690, U1906, U2020) cell lines. (B) Densitometric analysis of relative levels of protein expression in H23, H1299, U1810 and H661 cell lines. Cell lines distributed according to radiosensitivity, measured as the fraction surviving at 2 Gy (SF2). Equal loading was verified using anti-β-actin antibodies. Results are representative of three independent experiments.</p

Depletion of a component of the RNA-induced silencing complex (RISC), Argonaute2, does not affect the radioresistance of NSCLC.

<p>(A) The level of Argonaute2 (AGO2) mRNA in U1810 cells transfected with control (scram) or AGO2 siRNA normalized against 18S ribosomal RNA. Results are the mean±S.E.M. of three independent experiments. (B) Cleavage of PARP and processing of caspase-3 and -9 in U1810 cells transfected (48 h) with control (si scr) or AGO2 siRNA, and then subjected to irradiation for 48 h. Equal loading was verified using anti-GAPDH antibodies. All data are representative of three independent experiments. (C) Detection of apoptotic cell death in U1810 cells after transfection (48 h) with control or AGO2 siRNA and subsequent treatment with irradiation (48 h). (D) Caspase-3-like activity (fold increase with respect to control) in U1810 cells after treatment with either irradiation alone or in combination with transfection with control or AGO2 siRNA. All results shown are the mean±S.E.M. of three independent experiments.</p

Cardioprotective effect of 5-lipoxygenase gene (ALOX5) silencing in ischemia-reperfusion

It is well known that 5-lipoxygenase derivates of arachidonic acid play an important pathogenic role during myocardial infarction. Therefore, the gene encoding arachidonate 5-lipoxygenase (ALOX5) appears to be an attractive target for RNA interference (RNAi) application. In experiments on cultivated cardiomyocytes with anoxia-reoxygenation (AR) and in vivo using rat model of heart ischemia-reperfusion (IR) we determined influence of ALOX5 silencing on myocardial cell death. ALOX5 silencing was quantified using real-time PCR, semi-quantitative PCR, and evaluation of LTC4 concentration in cardiac tissue. A 4.7-fold decrease of ALOX5 expression (P < 0.05) was observed in isolated cardiomyocytes together with a reduced number of necrotic cardiomyocytes (P < 0.05), increased number live (P < 0.05) and unchanged number of apoptotic cells during AR of cardiomyocytes. Downregulation of ALOX5 expression in myocardial tissue by 19% (P < 0.05) resulted in a 3.8-fold reduction of infarct size in an open chest rat model of heart IR (P < 0.05). Thus, RNAi targeting of ALOX5 protects heart cells against IR injury both in culture and in vivo