The neuro-steroid, 3beta androstene 17alpha diol exhibits potent cytotoxic effects on human malignant glioma and lymphoma cells through different programmed cell death pathways

Abstract

The neuro-steroids 3beta-androstene-17alpha-diol (17alpha-AED), 3beta-androstene-17beta-diol (17beta-AED), 3beta-androstene-7alpha,-17beta-triol (7alpha-AET) and 3beta-androstene-7beta,-17beta-triol (7beta-AET) are metabolites of dehydroepiandrosterone and are produced in neuro-ectodermal tissue. Both epimers of androstenediols (17alpha-AED and 17beta-AED) and androstenetriols (7alpha-AET and 7beta-AET) have markedly different biological functions of their chemical analogue. We investigated the cytotoxic activity of these neuro-steroids on human T98G and U251MG glioblastoma and U937 lymphoma cells. Proliferation studies showed that 17alpha-AED is the most potent inhibitor, with an IC(50) approximately 15 microM. For T98G glioma, 90% inhibition was achieved with 25 muM of 17alpha-AED. Other neuro-steroids tested only marginally suppressed cell proliferation. Reduced cell adherence and viability could be detected after 18 h of 17alpha-AED exposure. Treatment with 17alpha-AED induced a significant level of apoptosis in U937 lymphoma cells, but not in the glioma cells. Cytopathology of 17alpha-AED-treated T98G cells revealed the presence of multiple cytoplasmic vacuoles. Acridine orange staining demonstrated the formation of acidic vesicular organelles in 17alpha-AED-treated T98G and U251MG, which was inhibited by bafilomycin A1. These findings indicate that 17alpha-AED bears the most potent cytotoxic activity of the neuro-steroids tested, and the effectiveness may depend on the number of hydroxyls and their position on the androstene molecule. These cytotoxic effects may utilize a non-apoptotic pathway in malignant glioma cells.

Figure 1

The molecular structure of 17α-AED, 17β-AED, 7α-AET and 7β-AET androstene neuro-steroids.

Figure 2

17α-AED is a potent inhibitor of tumour cell proliferation. (A) T98G glioma cells, (B) U251MG glioma cells and (C) U937 lymphoma cells were cultured for 3 days with titrated doses of 17α-AED (•); 17β-AED (○); 7α-AET (▪) or 7β-AET (□), in a standard proliferation assay. Incorporation of ³H-TdR was used as a measure of cell proliferation, and the percentage in which proliferation was inhibited in the androstene neuro-steroid-treated cultures as compared to sham-treated cultures is indicated. A representative experiment of at least two independent experiments is shown.

Figure 3

Cytotoxic effects of 17α-AED. Tumour cells were seeded onto culture dishes and incubated for ∼8 h before the addition of 17α-AED. After 18 h of treatment, cultures were photographed and the viability of cells was determined. (A, C) T98G cells treated with vehicle or (B, D) 10 μM 17α-AED. (E) Cell death of T98G glioma and U937 lymphoma cells treated with 10 μM 17α-AED and U251MG glioma cells treated with 15 μM 17α-AED. Mean values of at least two independent experiments are shown. ^*P=0.001, ^**P⩽0.01.

Figure 4

17α-AED treatment induces DNA fragmentation of U937 lymphoma cells. (A) U937 lymphoma cells; (B) T98G glioma cells and (C) U251MG glioma cells were treated with vehicle (left panels) or 17α-AED (right panels). After 4 days, the degree of DNA cleavage was assessed by TUNEL staining and used as a measure of apoptosis. Annotated numbers reflect the percentage of cells staining positive for apoptosis (x-axis, FITC-A). This experiment was performed twice and yielded similar results.

Figure 5

Treatment with 17α-AED induces cleavage of caspase 3 and PARP in U937 lymphoma cells, but not in T98G and U251MG glioma cells. Whole-cell lysates were obtained from human U937, T98G and U251MG cells treated for 3 days with vehicle or 17α-AED (10 μM). A 30 μg weight of total protein from these cells were subjected to Western blot analysis, and probed with an Ab specific for the total caspase 3 or PARP. A reduced level of pro-caspase 3 or PARP, accompanied by the presence of their smaller cleavage products can be seen 17α-AED-treated U937 cells as compared to cells treated with vehicle, but not in treated malignant glioma cells. An anti-β-actin mAb was used to show equal protein loading.

Figure 6

Cytopathology of T98G cells exposed to vehicle or 17α-AED (10 μM) for 30 h. (A) T98G cultures treated with vehicle are composed of cells with epithelioid morphology; a large, pale staining nucleus with prominent nucleoli. A T98G cell undergoing mitosis (M). (B, C) Numerous cells in the 17α-AED-treated cultures show the presence of multiple cytoplasmic vacuoles (arrows) and small, basophilic bodies (arrow heads). Haematoxylin and eosin staining, × 100 magnification with oil.

Figure 7

Acridine orange staining of 17α-AED-treated tumour cells indicates a high level of acidic vacuoles in T98G glioma cells, but not in U937 lymphoma cells. (A) Treatment with 10 μM of 17α-AED (middle) induces a high level of AVO formation in T98G cells as compared to T98G cells treated with vehicle (left). The presence of bafilomycin A1 (baf., 50 nM) inhibits 17α-AED (10 μM)-induced AVO formation in T98G cells (right). (B) A low level of acidic vacuoles can be detected in U937 cells treated with 10 μM 17α-AED (right) as compared to sham-treated cells (left). Annotated numbers indicate the percentage of cells positive for acidic vacuoles, quadrant Q2. Representative results of at least three independent experiments are shown.