Onconase mediated NFKβ downregulation in malignant pleural mesothelioma

Our plain-English summary of Onconase mediated NFKβ downregulation in malignant pleural mesothelioma, by C.M. Goparaju, J.D. Blasberg, S. Volinia, et al (Oncogene, 2011).

The study

• Authors: C.M. Goparaju, J.D. Blasberg, S. Volinia, J. Palatini, S. Ivanov, J.S. Donington, C. Croce, M. Carbone, H. Yang, H.I. Pass
• Journal: Oncogene
• Published: 2011;30(24):2767-77
• Cited by: 52 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Treatment of malignant pleural mesothelioma (MPM) with Ranpirnase (Onconase) results in disruption of protein translation and cell apoptosis. We hypothesize that Onconase exerts an effect via downregulation of nuclear factor kappa B (NFKβ) by specific microRNAs (miRNAs) and that interference of this pathway could have implications for MPM resistance to chemotherapy. Three immortalized MPM cell lines (H2959, H2373 and H2591) were exposed to Onconase at 0-20 μg/ml. Cell counts were measured at 48 and 72 h. Gene expression in miRNA-enriched RNA was validated by reverse transcription-PCR (RT-PCR). The functional implications of miRNA expression were evaluated by transfecting miRNA mimics or inhibitors into MPM cell lines, and performing Matrigel invasion, cell proliferation, soft agar colony formation and scratch closure assays. Effects on NFKβ expression and downstream targets including ABC transporters, BCL-xl and IAP were assessed by RT-PCR and western blotting. Treatment with 20 μg/ml of Onconase significantly decreased cell count and invasion. Hsa-miR-17* was significantly upregulated and hsa-miR-30c was significantly downregulated by Onconase treatment in all cell lines. Forced expression of hsa-miR-17* mimic and hsa-miR-30c inhibitor each significantly decreased functional activity of Onconase in all assays. NFKB1 (p50) expression and downstream targets were also decreased with Onconase treatment, as well as with forced expression of miRNA mimic and inhibitors. Onconase treatment caused a significant decrease in cell proliferation, invasion and in expression of certain miRNAs. Recapitulation of the resultant miRNA expression pattern with hsa-miR-17* mimic and hsa-miR-30c inhibitor resulted in downregulation of NFKB1 and reduced malignant behavior in functional assays. Thus, Onconase likely exerts its antitumor effect through these miRNAs.

In plain English

This was a laboratory study of mesothelioma cells grown in dishes, not of people. The authors used three malignant pleural mesothelioma cell lines established in their laboratory from surgical specimens. They treated the cells with the drug Onconase (ranpirnase) at doses up to 20 µg/mL for 48 and 72 hours. Asbestos exposure was not studied. At 20 µg/mL, Onconase significantly reduced cell growth and cell invasion in all three lines. It also changed the levels of several microRNAs, which are small RNA molecules that regulate genes. On array testing, hsa-miR-17* rose 30-fold and hsa-miR-30c fell 41% compared with untreated cells. When the authors artificially raised miR-17* or blocked miR-30c in the cells, invasion, colony growth in soft agar and proliferation all fell. For example, blocking miR-30c reduced invasion by 81 to 96%. These changes also lowered levels of NFKB1 and of the multidrug resistance gene ABCB1. A reporter test in HEK 293T cells suggested that miR-17* acts directly on NFKB. The authors concluded that Onconase likely exerts its anti-tumor effect through these microRNAs.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) experiment on human mesothelioma cell lines, with microRNA arrays, RT-PCR, Western blotting, transfection of microRNA mimics and inhibitors, functional assays, and luciferase reporter assays
• Who was studied: Three malignant pleural mesothelioma cell lines, named H2373, H2591 and H2595 in the methods (H2959 in the abstract), established from surgical specimens in the authors' laboratory. HEK 293T cells were used for the reporter assays.
• Exposure: Onconase (ranpirnase) at 0, 1.25, 2.5, 5, 10 or 20 µg/mL for 48 and 72 hours, and transfection with an hsa-miR-17* mimic and an hsa-miR-30c inhibitor. No asbestos exposure was studied.
• Main results:
  • In all three cell lines, 20 µg/mL Onconase significantly decreased cell proliferation at 48 and 72 hours. Untreated control cells increased 2-fold in number at 48 hours and 3-fold at 72 hours.
  • In the three cell lines, 20 µg/mL Onconase significantly reduced invasive capacity.
  • On microRNA arrays, 20 µg/mL Onconase for 72 hours raised hsa-miR-17* by 30-fold and lowered hsa-miR-30c by 41% compared with controls.
  • By RT-PCR, 20 µg/mL Onconase raised hsa-miR-17* 2.46 to 3.23-fold, and hsa-miR-30c fell 61 to 63% after Onconase treatment.
  • Compared with controls, the hsa-miR-17* mimic decreased cell invasion by 31 to 48% (p less than or equal to 0.004) and the hsa-miR-30c inhibitor by 81 to 96% (p less than or equal to 0.001), in all cell lines.
  • Anchorage-independent (soft agar) growth fell 31 to 47% with the miR-17* mimic and 32 to 46% with the miR-30c inhibitor. Cell number fell 25 to 33% with the mimic and 35 to 80% with the inhibitor.
  • The miR-17* mimic and the miR-30c inhibitor reduced expression of ABCB1 and NFKB1 compared with controls. This was confirmed by Western blotting.
  • In HEK 293T cells, the hsa-miR-17* mimic decreased NFKβ luciferase activity to 58% of the negative control (p = 0.004). The hsa-miR-30c mimic had no effect. Neither microRNA directly affected the ABCB1 reporter.
  • The mimic and inhibitor decreased expression of BCL-xL, cIAP1 and MMP9.
  • The reverse experiment, inhibiting miR-17* and raising miR-30c, reversed the cell invasion results.
• The authors' conclusion: The authors concluded that Onconase likely exerts its anti-tumor effect through hsa-miR-17* and hsa-miR-30c, which they believe are intermediates in an effect on NFKβ.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Apoptosis, Nuclear Factor kappa B (NF-κB), Chemotherapy.

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