Fig 1.
K+ current-dependent cytotoxicity of LaFr26 and oxyopinin-2b.
(A and B) LaFr26 (A) and oxyopinin-2b (B) were added to the media of 293T cells that stably expressed Kir2.1 at indicated concentrations in the presence (Ba2+) or absence (w/o Ba2+) of 0.3 mM BaCl2. Cell viabilities were measured 30 min after addition (Student’s t-test, Ba2+ vs w/o Ba2+, n = 4).
Fig 2.
Resting membrane potential-dependency of cytotoxicity of the venom peptides.
(A) LaFr26 was added to the media of 293T cells that stably express GFP, HERG-1, TREK-1, or Kir2.1, and cell viabilities were measured (ANOVA followed Student’s t-test, vs 0 μM, n = 6). (B) A significant correlation was found between cell viabilities at 10 μM and the resting membrane potential measured with whole-cell patch-clamp recordings. (C) Oxyopinin-2b was added to the media of these cells (n = 6). (D) Cell viability at 10 μM again correlated with the resting membrane potentials.
Fig 3.
Endogenous expression of TREK-like current in the lung cancer LX22 and BEN cells.
After whole-cell access was made from LX22 and BEN cells, outward rectified currents were evoked by the step pulses indicated as protocol (E). (A and B) LX22 cells expressed currents resistant to 5 μM ML365 and sensitive to bupivacaine, which were similar to TREK-1 current. (C and D) BEN cells also expressed 0.3 mM bupivacaine-sensitive current. (F and G) I-V relationship of the whole cell current expressed in LX22 and BEN cells. The I-V relationship indicates the outward rectification of the current and sensitivity to bupivacaine.
Fig 4.
Cytotoxicity of LaFr26 and oxyopinin-2b to LX22 and BEN cells, but not to U87 or T98G cells.
(A and B) LaFr26 and oxyopinin-2b were added to the media of LX22 cells, and the cell viabilities were measured 30 min after (ANOVA followed Student’s t-test, vs 0 μM, n = 6). (C) LX22 cells were incubated with these peptides (10 μM) in the presence or absence of a K+ channel blocker, BaCl2 (1 mM) and viability were measured. The addition of Ba2+ inhibited the cytotoxicity of peptides, indicating the K+ channel current dependency. (D and E) LaFr26 and oxyopinin-2b were added to the media of BEN cells (n = 6). (F) The addition of Ba2+ again inhibited the cytotoxicity to BEN cells. (G, H, and I) LaFr26 and oxyopinin-2b were added to the media of U87 or T98G cells. No cytotoxicity was observed. (J) Resting membrane potentials of U87, T98G, LX22, and BEN cells (n = 10, 5, 7, and 5).
Fig 5.
Schematic illustration of the designed lentiviral vectors used to transduce the cells with the genetic constructions for the expression of LaFr26, oxyopinin-2b, and mCherry.
Lv-LaFr26 (A) and Lv-Oxy-2b (B) carry IRES for the bicistronic coexpression of GFP and the spider peptides, which were designed to be secreted byLaFr26 or oxyopinin-2b. (C) Structure of Lv-mCherry. The expression cassettes were inserted in the downstream of RNA packaging signal between two long terminal repeats. (β-actin, promoter of chick β-actin used for the coexpression; GFP, green fluorescent protein gene; IRES, internal ribosomal entry site for the bicistronic coexpression; Signal, Gaussia luciferase signal peptide; mCherry, mCherry gene) (D) Schematic illustration of the autotoxic expression of the venom peptides with Lv-LaFr26 and Lv-Oxy-2b.
Fig 6.
K+ channel current-dependent cytotoxicity Lv-LaFr26- and Lv-Oxy-2b-transduced cells.
(A, B, and C) Lv-LaFr26, Lv-Oxy-2b, and Lv-mCherry were added to the media of 293T cells that stably express GFP or Kir2.1 and the cell viabilities were measured 72 h after addition. Both Lv-LaFr26 and Lv-Oxy-2b transductions resulted in cytotoxicity only to Kir2.1-expressing cells (A and B). (C) Contrastingly, Lv-mCherry infection did not lead to any cytotoxicity to GFP- or Kir2.1-expressing cells (n = 4). (D) 56–3 cells were incubated with conditioned media from the cells transduced with Lv-mCherry, Lv-LaFr26, and Lv-Oxy-2b. Cell viabilities were measured after 24 h (n = 8, 4, and 4).
Fig 7.
Chromatogram of media of control vector and Lv-LaFr26 transduced cells.
(A and B) The supernatant (100 μL) of media collected 48 h after viral transduction were fractionated by a cation-exchange column. Ordinate indicates the A230 nm (arbitral unit of HPLC UV-detector) and NaCl concentration (mM). (C, D, E, F) Chromatogram from 15 to 25 min was enlarged.
Fig 8.
Decrease in the whole-cell membrane resistance in Lv-LaFr26 and Lv-Oxy-2b transduced cells.
(A) Whole-cell membrane resistance was measured 48 h after transduction. Both Lv-LaFr26 and Lv-Oxy-2b decreased the resistance compared to that with Lv-GFP (n = 9, 9, and 6, *, p < 0.05 vs Lv-GFP treatment). (B) There was no difference in access resistance.
Fig 9.
Cytotoxicity of Lv-LaFr26 and Lv-Oxy-2b to lung cancer cells.
(A, B, and C) Lv-LaFr26 and Lv-Oxy-2b were added to the media of lung cancer cell lines, LX22 and BEN. The cell viabilities decreased (n = 4).