Preferential radiosensitization to glioblastoma cancer stem cell-like cells by a Hsp90 inhibitor, N-vinylpyrrolidone-AUY922

Abstract

The present study examined the radiosensitization induced by a heat shock protein 90 inhibitor, N-vinylpyrrolidone (NVP)-AUY922, in CD133-positive cells in a hypoxic area of T98G spheroids. CD133-positive cells that are induced in the hypoxic microenvironment of spheroids have previously been reported to exhibit cancer stem cell-like properties. The present study used CD133-positive cells from a glioblastoma cell line (T98G) as cancer stem cell-like cells. CD133-positive and negative cells were sorted from T98G spheroids using fluorescence-activated cell sorting and used for colony formation assay. Colony formation assay results indicated that NVP-AUY922 enhanced radiosensitivity more strongly in CD133-positive cells compared with CD133-negative cells. This result showed that NVP-AUY922 was a preferential radiosensitization candidate targeting glioblastoma cancer stem cells. The mechanisms underlying radiosensitization by NVP-AUY922 are discussed in relation to the properties of cancer stem cells. Overall, HIF-1α inhibition by NVP-AUY922 may induce higher sensitization of cancer stem cells to radiation.

Keywords: N-vinylpyrrolidone-AUY922; cancer stem cell; glioblastoma; heat shock protein 90 inhibitor; radiosensitization.

Figure 1.

A schema on significance of CD-133 positive cells. The figure shows cancer stem cell-like properties in CD133-positive cells in T98G cell line-derived spheroids.

Figure 2.

Images of CD133/HIF-1α immunofluorescent double staining. Monolayer cultured cells staining for (A) CD133, (B) HIF-1α and (C) CD133/HIF-1α; spheroid sections on day 10 after cell seeding for (D) CD133 (red-colored), (E) HIF-1α (green-colored) and (F) CD133/HIF-1α (yellow-colored). Images of CD133/nestin immunofluorescence double staining: Monolayer cultured cells staining for (G) CD133, (H) nestin and (I) CD133/nestin; spheroid sections on day 10 after cell seeding for (J) CD133 (red-colored), (K) nestin (green-colored) and (L) CD133/nestin (yellow-colored). HIF, hypoxia-inducible factor-1α.

Figure 3.

Gating of CD133-positive and CD133-negative cells. Histograms on PE (labeled to CD133 antibody) signals of (A) monolayer-cultured cells and (B) spheroid-cultured cells. Cell distributions based on PE and 7-AAD signals from (C) monolayer-cultured cells and (D) spheroid-cultured cells. Spheroid or monolayer CD133-positive cells (red-colored) and spheroid CD133-negative cells (blue-colored) were sorted from the gating shown in (C and D), respectively. The gating was set on the basis of microscopic observation of PE signals in living cells (7-AAD-negative cells). 7-AAD, 7-aminoactinomycin D.

Figure 4.

Surviving fractions of SCPCs and SCNCs. SCPCs (CD133⁺; black columns) and SCNCs (CD133⁻; white columns) sorted from T98G spheroids were irradiated with X-rays, treated with N-vinylpyrrolidone-AUY922 alone or combined with X-rays. All plots represent the means and standard deviations of at least three independent experiments. *P<0.01 (Tukey's HSD test); ^#P<0.01 (Student's t-test). SCPCs, spheroid CD133-positive cells; SCNCs, spheroid CD133-negative cells.