IFN-γ restores the impaired function of RNase L and induces mitochondria-mediated apoptosis in lung cancer

Abstract

RNase L is an essential component in interferon (IFN)-mediated antiviral signaling that showed antitumor effects in cancer. Cancer immunotherapy based on interferon has achieved encouraging results that indicate an applicable potential for cancer therapy. Here we showed that function of RNase L, though highly upregulated, was functionally impaired both in nuclear and cytoplasm in lung cancer cells. In normal lung epithelial cells, RNase L activation induced by 2-5A promoted nuclear condensation, DNA cleavage, and cell apoptosis, while in lung cancer cells, these processes were inhibited and RNase L-mediated downregulation of fibrillarin, Topo I and hnRNP A1 was also impaired in lung cancer cells. Moreover, the impairment of RNase L in lung cancer cells was due to the elevated expression of RLI. Application of IFN-γ to lung cancer cells led to enhanced expression of RNase L that compensated the RLI inhibition and restored the cytoplasmic and nuclear function of RNase L, leading to apoptosis of lung cancer cells. Thus, the present study discovered the impaired function and mechanism of RNase L in lung cancer cells and proved the efficacy of IFN-γ in restoring RNase L function and inducing apoptosis in the lung cancer cell. These results indicated the RNase L as a therapeutic target in lung cancer cells and immunotherapy of IFN-γ may serve as an adjuvant to enhance the efficacy.

Fig. 1. Elevated expression with impaired cleavage activity of RNase L in lung cancer cells.

a Immunocytochemistry and quantitative analysis of RNase L in normal pulmonary epithelial cells (BEAS-2B), lung cancer cell (NCL-H157, GLC-82, Calu-3, LTEP-s, NCL-H520, NCL-H209, PG-LH7), and breast cancer cell (T47D). b–d Western blot of the total (b), cytoplasmic (c), and nuclear (d) RNase L in the indicated cell lines. e Cleavage activity to 28S rRNA in the indicated time points of RNase L in BEAS-2B cells (n = 3). f: Cleavage activity to 28S rRNA at 24 h of RNase L in lung cancer cell lines and breast cancer cells (T47D) (n = 3). ^♦p < 0.05; ^♦♦p < 0.01; ^♦♦♦p < 0.001, Student’s t test

Fig. 2. RNase L functions in the nucleus and induces nuclear condensation and DNA cleavage in lung epithelial cells.

Immunocytofluorescence of RNase L and DAPI in BEAS-2B and NCI-H157 stimulated by 2–5A at the indicated time points. b Immunocytofluorescence of TUNEL and DAPI in BEAS-2B and NCI-H157 stimulated by 2–5A at the indicated time points. c Western blot of RNase L in BEAS-2B interfered with si-RNase L or not. d Immunocytofluorescence of RNase L, DAPI, and Fibrillarin in BEAS-2B interfered with si-RNase L or not. e Western blot of fibrillarin, Topo I, and hnRNP A1 in the nucleus or cytoplasmic fractions; Lamin B and GAPDH are regarded as the internal reference of n nucleus and cytoplasmic fractions, respectively

Fig. 3. Suppressed dimerization of RNase L in lung cancer cells due to interaction with RLI.

a, c Western blot of monomer and dimer of RNase L in the nucleus or cytoplasmic of BEAS-2B and NCI-H157 cells. b, d Western blot of RLI and IP of RNase L and RLI in the nucleus and cytoplasm in BEAS-2B and NCI-H157. e Western blot of RLI and RNase L in total cell lysate, fibrillarin, Topo I, and hnRNP A1 in the nucleus or cytosolic fractions after the interference of RLI (si-RLI)

Fig. 4. Upregulation of RNase L by IFN-γ neutralized RLI and restored cleavage activity.

a, b Dose dependence (a) and time course (b) of IFN-γ in restoring the cleavage activity of RNase L (n = 3). c Cleavage activity of RNase L in the presence of IFN-γ in BEAS-2B and NCI-H157 cells (n = 3). d Western blot of OAS1, OAS2, and OAS3 in the presence of IFN-γ in BEAS-2B and NCI-H157 cells. e, f Western blot verification and rRNA cleavage after OAS3 interference by siRNA; g, j Western blot of RNase L dimer or monomer in the cytoplasm (g) or nucleus (j) in the presence of IFN-γ in BEAS-2B and NCI-H157 cells. h, i, k, l Cytoplasmic (h, i) and nuclear (k, l) RLI expression (i, l) and IP with RNase L (h, k) in the presence of IFN-γ in BEAS-2B and NCI-H157 cells

Fig. 5. IFN-γ promoted apoptosis activated by RNase L.

a, b Western blot of OAS1, OAS2, OAS3, RNase L, cytochrome C, and Caspase-9 in the mitochondrial or cytosolic fraction, co-immunoprecipitation of Bax and Bak, and cleavage activity of RNase L in NCl-H157 cells after the interference of RNase L. Prohibitin and GAPDH were regarded as the internal reference of mitochondria or cytosolic fractions, respectively. c Mitochondrial or cytosolic efflux of cytochrome C at different time points of IFN-γ stimulation (n = 3). d Western blot of Caspase-9, Caspase-3, and PARP in BEAS-2B and NCI-H157 cells stimulated with IFN-γ at different time points (n = 3). e, f Western blot of Caspase-9, Caspase-3, and PARP in BEAS-2B (e) and NCl-H157 (f) cells stimulated with IFN-γ and Caspase-9 inhibitor (z-LEHD-fmk) or Caspase-3 inhibitor (z-DEVD-fmk) (n = 3). g Accumulated DNA ladder after IFN-γ stimulation in BEAS-2B and NCI-H157 cells (n = 3)

Fig. 6

Graphical abstract of the mechanism of IFN-γ promoting the RNase L function