Fig 2: SENP1-induced de-SUMOylation causes remarkable downregulation of NPM-ALK protein expression. (A) Karpas 299 and SR-786 cells were transfected with EV or Myc-SENP1 expression plasmid. Myc tag was used to distinguish transfected SENP1 from the very low levels of endogenously expressed SENP1 protein. Subcellular fractionation shows that in EV-transfected cells, SUMO-1 and SUMO-2/3 proteins are exclusively localized in the nuclear (N) compartment with lack of expression in the cytoplasm (C). Although the NPM-ALK protein is distributed between the nucleus and cytoplasm, it is slightly more abundant in the nucleus. It is important to mention that similar patterns of expression of the SUMO modifiers as well as NPM-ALK were seen in Karpas 299 and SR-786 cells under the basal conditions without exposing the cells to the transfection stress (data not shown), supporting that these patterns of expression are physiological. Notably, transfection of SENP1 induced remarkable downregulation of SUMO-1 and SUMO-2/3 proteins in the two cell lines. The decrease in SUMO modifiers was associated with marked decrease in the nuclear and cytoplasmic expression of NPM-ALK. Lamin A/C and β-actin were used as nuclear and cytoplasmic loading controls, respectively. (B) To examine whether adequate transfection was achieved, immunofluorescence staining for Myc-SENP1 was performed, and its levels of expression were monitored for up to 48 hours. Transfection of EV shows total lack of SENP1. Similarly, SENP1 was not detected either in Karpas 299 or in SR-786 cells immediately after transfection at baseline. After 15 minutes, low levels of expression of SENP1 were identified in the cytoplasm. At 30/60 minutes, the cytoplasmic expression of SENP1 became much more pronounced. Gradual shuttling of SENP1 into the nucleus occurred, and it became distributed between the nucleus and cytoplasm at 3/6 hours. However, at 24 hours, most of SENP1 appeared to be shuttling back to the cytoplasm, and yet at 48 hours, much higher levels of SENP1 were distributed between the nucleus and cytoplasm.

Fig 3: Proposed model for NPM-ALK SUMOylation. NPM-ALK forms heterodimers with wild-type NPM. These NPM/NPM-ALK heterodimers possess ability to shuttle between the nucleus and cytoplasm. The association between NPM/NPM-ALK, through its Lys24 and Lys32 residues, with the SUMO proteins occurs predominantly in the nucleus, which is the cellular compartment where SUMO proteins appear to be predominantly expressed in NPM-ALK+ T-cell lymphoma cells. In turn, conjugation with the SUMO modifiers confers stability on NPM-ALK oncogenic protein by preventing its ubiquitination and degradation. Therefore, NPM/NPM-ALK is capable of shuttling back to the cytoplasm. To this end, SUMOylation-mediated stability leads to accumulation and abundant expression of NPM-ALK in the nucleus and cytoplasm. At the other hand, SENP1-mediated de-SUMOylation decreases significantly the expression and accumulation of NPM/NPM-ALK in the nucleus and cytoplasm through the increase in NPM-ALK breakdown that occurs through the switch from the protective effects of SUMOylation to the ubiquitination-induced degradation.

Fig 4: Effect of Senp1-overexpression on H2O2 in HL-1 cells. (A) The effects of different concentrations of AS-IV (25, 50, 100 or 200 µmol/l) on the survival of HL-1 cells subjected to ISO. One-way ANOVA, followed by Tukey's test. (B) The expression of Senp1 protein was examined by Western blotting following HA-Senp1 plasmid transfection. T-test. (C) Compared with the control (dimethyl sulfoxide, 0.1%), H2O2 was increased in the 20 µmol/l ISO-induced HL-1 cells. AS-IV (50 µmol/l, n=6) prevented the ISO-induced increase in H2O2, which was inhibited by Senp1-overexpression (n=7). Data are presented as the mean ± standard deviation. Two-way ANOVA followed by Bonferroni's test. *P<0.05 vs. control; #P<0.05 vs. HF; &P<0.05 vs. HF+AS-IV. H2O2, hydrogen peroxide; AS-IV, astragaloside; ANOVA, analysis of variance; ISO, isoprenaline; Senp1, small ubiquitin-like modifier-specific protease 1.

Fig 5: (A) TUNEL staining was used to examine cell apoptosis in HL-1 cells. Compared with the control (dimethyl sulfoxide, 0.1%), cell apoptosis was increased in 20 µmol/l ISO-induced HL-1 cells. AS-IV (50 µmol/l, n=10) prevented the ISO-induced increase in cell apoptosis, which was inhibited by Senp1-overexpression. (B) Summarized data of the cell apoptotic rate (n=7). Two-way analysis of variance followed by Bonferroni's test. Data are presented as the mean ± standard deviation. *P<0.05 vs. control; #P<0.05 vs. HF; &P<0.05 vs. HF+AS-IV. ISO, isoprenaline; AS-IV, astragaloside; Senp1, small ubiquitin-like modifier-specific protease 1.