Growth of a human mammary tumor cell line is blocked by galangin, a naturally occurring bioflavonoid, and is accompanied by down-regulation of cyclins D3, E, and A

Abstract

Introduction: This study was designed to determine if and how a non-toxic, naturally occurring bioflavonoid, galangin, affects proliferation of human mammary tumor cells. Our previous studies demonstrated that, in other cell types, galangin is a potent inhibitor of the aryl hydrocarbon receptor (AhR), an environmental carcinogen-responsive transcription factor implicated in mammary tumor initiation and growth control. Because some current breast cancer therapeutics are ineffective in estrogen receptor (ER) negative tumors and since the AhR may be involved in breast cancer proliferation, the effects of galangin on the proliferation of an ER-, AhRhigh line, Hs578T, were studied.

Methods: AhR expression and function in the presence or absence of galangin, a second AhR inhibitor, alpha-naphthoflavone (alpha-NF), an AhR agonist, indole-3-carbinol, and a transfected AhR repressor-encoding plasmid (FhAhRR) were studied in Hs578T cells by western blotting for nuclear (for instance, constitutively activated) AhR and by transfection of an AhR-driven reporter construct, pGudLuc. The effects of these agents on cell proliferation were studied by 3H-thymidine incorporation and by flow cytometry. The effects on cyclins implicated in mammary tumorigenesis were evaluated by western blotting.

Results: Hs578T cells were shown to express high levels of constitutively active AhR. Constitutive and environmental chemical-induced AhR activity was profoundly suppressed by galangin as was cell proliferation. However, the failure of alpha-NF or FhAhRR transfection to block proliferation indicated that galangin-mediated AhR inhibition was either insufficient or unrelated to its ability to significantly block cell proliferation at therapeutically relevant doses (IC50 = 11 microM). Galangin inhibited transition of cells from the G0/G1 to the S phases of cell growth, likely through the nearly total elimination of cyclin D3. Expression of cyclins A and E was also suppressed.

Conclusion: Galangin is a strong inhibitor of Hs578T cell proliferation that likely mediates this effect through a relatively unique mechanism, suppression of cyclin D3, and not through the AhR. The results suggest that this non-toxic bioflavonoid may be useful as a chemotherapeutic, particularly in combination with agents that target other components of the tumor cell cycle and in situations where estrogen receptor-specific therapeutics are ineffective.

Figure 1

Hs578T cells express nuclear aryl hydrocarbon receptor (AhR). Cytoplasmic and nuclear cell extracts prepared from subconfluent monolayers of malignant, estrogen receptor negative Hs578T cells were analyzed by western immunoblotting with AhR-specific antibody following SDS-PAGE. Blots were stripped and re-probed for lamin A/C and α-tubulin to confirm purity of the nuclear and cytoplasmic cell fractions, respectively. Representative data from a total of three experiments are shown.

Figure 2

Galangin inhibits aryl hydrocarbon receptor-dependent pGudLuc reporter activity. Hs578T cells were left untransfected or were transfected with 0.5 μg/well renilla luciferase vector phRL-TK and 0.1 μg control pGL3 or pGudLuc vector per well and treated with 10^-4 to 10^-5 M galangin, 10^-4 to 10^-5 M indole 3-carbinol (I3C), or 10^-6 M α-naphthoflavone (α-NF) in the (a) absence or (b) presence of 10^-9 M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cells were harvested 18 hours later and luciferase activity assayed. Firefly luciferase activity was normalized to renilla activity in each experiment. (a) Data pooled from 4 to 16 experiments are presented as the average fold increase relative to non-transfected cells + standard error. An asterisk (*) indicates a significant difference relative to vehicle-treated controls, p < 0.02. A cross (+) indicates p = 0.056. (b) Data pooled from 4 to 16 experiments are presented as the average fold increase relative to non-transfected cells + standard error. An asterisk (*) indicates a significant difference relative to vehicle-treated controls, p < 0.02. A hash sign (#) indicates a significant increase in activity relative to untreated, pGudLuc-transfected controls.

Figure 3

Galangin inhibits proliferation of Hs578T breast cancer cells. Hs578T cells were treated in triplicate with vehicle, 10^-4 to 10^-6 M galangin, 10^-4 to 10^-6 M indole 3-carbinol (I3C), or 10^-5-10^-6 M α-naphthoflavone (α-NF) and grown in 3H-thymidine-containing media for 18 hours. Triplicates were averaged for each point in each experiment. (a) Data are pooled from 3 to 11 experiments and presented as the percent of control (vehicle-treated) counts per minute (CPM) + standard error. In 11 experiments, the average CPM in vehicle-treated controls was 35,583 + 6,893. An asterisk (*) indicates a significant decrease in ³H-thymidine incorporation relative to vehicle controls, p < 0.05. (b) Data obtained with galangin as above were replotted to determine the IC₅₀ (median inhibition concentration) (median inhibition concentration). The calculated IC₅₀ was 11 μM.

Figure 4

Aryl hydrocarbon receptor (AhR) repressor (FhAhRR) inhibits AhR-dependent pGudLuc reporter activity. Hs578T cells were left untransfected or were transfected with 0.5 μg/well renilla luciferase vector phRL-TK and 0.1 μg pGudLuc/well together with 0.5 μg control vector (pcDNA), 0.1 μg FhAhRR, or 0.5 μg FhAhRR in the (a) absence or (b) presence of 10^-9 M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cells were harvested 18 hours later and luciferase activity assayed. Firefly luciferase activity was normalized to renilla activity in each experiment. Data pooled from six experiments are presented as the average fold increase relative to non-transfected cells + standard error. An asterisk (*) indicates a significant difference relative to pcDNA-transfected controls, p < 0.001.

Figure 5

Aryl hydrocarbon receptor repressor does not inhibit proliferation of Hs578T breast cancer cells. Hs578T cells were transfected with control pcDNA vector or with FhAhRR plasmid as in Figure 4, plated in triplicate in 96-well plates, and allowed to adhere overnight before addition of ³H-thymidine. Cells were harvested 18 hours later and assayed for ³H-thymidine incorporation. Triplicates were averaged in each experiment. Data are pooled from three experiments and are presented as the mean counts per minute (CPM) + standard error. There were no statistical differences between groups.

Figure 6

Galangin and indole 3-carbinol (I3C) block Hs578T cells progression from G₀/G₁ into cell cycle. Hs578T cells were synchronized by serum deprivation for 48 h followed by rescue with 10% serum. As indicated, 10^-4 to 10^-6 M galangin (Gal), 10^-5-0^-6 M α-naphthoflavone (α-NF), or 10^-4 M I3C were added to triplicate wells at the time of serum rescue. Cells were harvested 24 hours later and assayed for DNA content by propidium iodide (PI) staining and flow cytometry. Data from triplicate wells were averaged in each experiment. (a) Flow cytometry histograms from one representative experiment in which 10^-4 M galangin, 10^-5 M α-NF, and 10^-4 M I3C were used are presented. (b) Data pooled from 4 to 9 experiments are presented as the mean percentage of cells in G₀/G₁ + standard error. An asterisk (*) indicates a significant increase relative to serum starved, vehicle treated cultures, p < 0.01. A cross (+) indicates a significant increase relative to untreated cultures, p < 0.01.

Figure 7

Galangin down-regulates expression of cyclins D3, A, and E. Hs578T cells were left untreated or were treated with vehicle, 10^-4 M galangin, 2.5 × 10^-4 M indole 3-carbinol (I3C), or 10^-5 M α-naphthoflavone (α-NF) and assayed for cyclin D1, D3, E, and A expression 18 hours thereafter by western blotting. Blots were stripped and reprobed with β-actin-specific antibody to control for sample loading. (a) Data from one representative experiment from a total of three experiments are presented. (b) Cyclin band densities were normalized with β-actin band densities and then expressed as a percentage of β-actin normalized cyclin expression in untreated cultures. Data are pooled from three experiments and expressed as the percent of control of the respective normalized band densities + standard errors. An asterisk (*) indicates a significant decrease in cyclin expression relative to vehicle controls, p < 0.001. A cross (+) indicates a significant decrease, p < 0.02.