4-IBP, a sigma1 receptor agonist, decreases the migration of human cancer cells, including glioblastoma cells, in vitro and sensitizes them in vitro and in vivo to cytotoxic insults of proapoptotic and proautophagic drugs

Abstract

Although the molecular function of sigma receptors has not been fully defined and the natural ligand(s) is still not known, there is increasing evidence that these receptors and their ligands might play a significant role in cancer biology. 4-(N-benzylpiperidin-4-yl)-4-iodobenzamide (4-IBP), a selective sigma1 agonist, has been used to investigate whether this compound is able to modify: 1) in vitro the migration and proliferation of human cancer cells; 2) in vitro the sensitivity of human glioblastoma cells to cytotoxic drugs; and 3) in vivo in orthotopic glioblastoma and non-small cell lung carcinoma (NSCLC) models the survival of mice co-administered cytotoxic agents. 4-IBP has revealed weak antiproliferative effects on human U373-MG glioblastoma and C32 melanoma cells but induced marked concentration-dependent decreases in the growth of human A549 NSCLC and PC3 prostate cancer cells. The compound was also significantly antimigratory in all four cancer cell lines. This may result, at least in U373-MG cells, from modifications to the actin cytoskeleton. 4-IBP modified the sensitivity of U373-MG cells in vitro to proapoptotic lomustin and proautophagic temozolomide, and markedly decreased the expression of two proteins involved in drug resistance: glucosylceramide synthase and Rho guanine nucleotide dissociation inhibitor. In vivo, 4-IBP increased the antitumor effects of temozolomide and irinotecan in immunodeficient mice that were orthotopically grafted with invasive cancer cells.

Figure 1

Expression of the splice variants of σ₁ receptor mRNA (V1–V5) in four different human cancer cell lines, namely, melanoma (C32), refractory prostate (PC3), NSCLC (A549), and glioma (U373-MG) cells, as assessed by RT-PCR analyses. (A) Schematic representation of the five different splice variants of σ₁ receptor mRNA, with the exact sites targeted by two different pairs of primers (P1 and P2) used to investigate the expression patterns of the splice variants. CDS = mRNA-coding region. Primer positions are outlined by black (P1) and gray (P2) hatched boxes, respectively. (B and C) The detection of splice variants of σ₁ receptor mRNA by RT-PCR using primer pairs P1 and P2, respectively. (D) The detection of splice variants of σ₁ receptor mRNA by RT-PCR using primer pairs P2 and P1, respectively, and carried out on purified fragments isolated after PCR performed with “external primers” (PE1 and PE2). (B–D) First lane: 1-kb plus DNA size ladder; Ct nontemplate control (H₂O). White arrows indicate the positions of different amplified splice variants. (?) Nonspecified amplification fragments (for RT-PCR experimental details, see Materials and Methods section).

Figure 2

Evaluation of the antiproliferative and antimotility activities of 4-IBP in cancer cells. (A) Percentages of living cells assessed by the colorimetric MTT assay in four human cancer cell lines (U373-MG, C32, A549, and PC3). The data are shown as mean ± standard error with respect to the 4-IBP concentrations used (see x-axis). (B) Distribution of MRDO values obtained for C32 cells treated with increasing 4-IBP concentrations (see x-axis) and those left untreated (Ct). The data show individual values (dotted) and corresponding medians (horizontal bars). (C) Typical quantification experiment of the motility levels of individual cells by establishing the trajectory of each cell centroid and by quantifying the MRDO variable (i.e., maximum distance covered by a cell normalized by its observation time, expressed in µm/hr).

Figure 3

Evaluation of 4-IBP - induced effects on U373-MG glioblastoma cells. (A) Illustration of the morphologic variables used to evaluate the roundness of a cell (level of sphericity, elongation, and morphologic pattern) and its spread (area). (B and C) Illustration of actin cytoskeleton distribution in control U373-MG cells and in those treated with 1 nM 4-IBP for 7 hours. Fibrillar actin and globular actin appear as green and red fluorescence, respectively. (D and E) Effects of two concentrations of 4-IBP on fibrillar/globular actin ratios and cell motility levels computed over different time periods (as indicated on the x-axis). (F) 4-IBP (10 nM) did not significantly increase [Ca²⁺]_i in U373-MG GBM cells when compared to ionomycin.

Figure 4

(A and B) Therapeutic combination of 4-IBP and cytotoxic agents [(A) lomustin and (B) temozolomide] in a scratch wound assay in which colonization by human U373-MG GBM cells of an artificially inflicted wound in a subconfluent cell population was followed over time. (A) and (B) illustrate delay in the wound healing process when U373-MG cells were pretreated with 4-IBP [10 nM; duration exposure varied from 0 (no 4-IBP treatment) to 24 hours on the x-axis] before the cytotoxic insult with lomustin or temozolomide, respectively, used at 1 µM (open dots) or 10 µM (open squares) over 16 hours. Controls were U373-MG cells cultured in the absence of 4-IBP, lomustin, or temozolomide, and were arbitrarily normalized to 100% wound colonization after 24 hours of culture (open triangles in A and B). An additional control was performed for each treatment [i.e., lomustin or temozolomide added alone at 1 µM (black lozenge) or at 10 µM (white lozenge) for 16 hours to the cells, and 4-IBP at 10 nM for 7 and 24 hours of exposure (white triangles)]. (C and D) Evaluation of the levels of expression of total (black bars) and phosphorylated (open bars) p85-PI3K (C) and Akt (D) in U373-MG GBM cells left untreated (Ct) or treated with 10 nM 4-IBP for the periods indicated. The data are expressed as mean ± SEM. Assays were validated using serum-starved U373-MG treated with epidermal growth factor (200 ng/ml) for 10 minutes (C₊). (E) Western blot analyses of PARP cleavage and p53 expression in U373-MG cells left untreated (0) or treated with 10 nM 4-IBP for between 48 and 96 hours.

Figure 5

(A) The detection and quantification of acidic vesicular organelles by acridine orange staining using flow cytometry in human U373-MG GBM cells treated for 72 hours with increasing concentrations of 4-IBP. In acridine orange-stained cells, the cytoplasm and nucleus fluoresce green (y-axis), whereas acidic compartments fluoresce red (x-axis). The intensity of the red fluorescence is proportional to the degree of acidity and to the volume of acidic vesicular organelles, including autophagic vacuoles. The values in (A) refer to the percentages of cells with a significant proportion of acidic vesicular organelles. (B–F) Western blot analyses of Beclin-1 (B); microtubule-associated protein 1 LC3 (an autophagosomal orthologue of yeast Atg8/Aup7) type I (LC3-I) and type II (LC3-II) (C); Hsp70 (D); GRP78 (E); and ORP150 (F) in human U373-MG glioblastoma cells treated with increasing concentrations of 4-IBP for 72 hours.

Figure 6

Immunofluorescence analyses in the investigation of the expression of Rho GDI (A–C) and GCS (D–F) in U373-MG GBM cells left untreated (A and D) or treated for 72 hours with 1 nM 4-IBP (B and E) and 10 nM 4-IBP (C and F). The levels of expression of Rho GDI and GCS are shown as red fluorescence (right panels), with bright-field microscope analysis (left panels) as morphologic control.

Figure 7

(A) The survival of mice orthotopically (brain) grafted with human U373-MG GBM cells after treatment with 4-IBP alone (red line) or combined with temozolomide (purple line), compared to temozolomide alone (green line) and controls (blue line). (B) The survival of mice orthotopically (lung) grafted with human A549 NSCLC cells after treatment with 4-IBP alone (red line) or combined with IRI (purple line), compared to IRI alone (green line) and controls (blue line).