Figure 1.
Gltn impairs the proliferation of triple-negative breast cancer cells in vitro.
(A) Triple-negative and luminal breast cancer cells were grown with Gltn or PBS as a vehicle control for 5 days at the indicated dose. Cell numbers were determined using trypan blue exclusion and hemacytometer in n = />3 experiments +/- S.E.* denotes significant differences compared to controls with p values = /< 0.05 (B) Sigmoidal dose response curves comparing Gltn sensitivity in luminal MCF-7 cells with triple-negative MDA-MB-468 cells. Horizontal bars on curves represent points of IC50. (C) Triple-negative cell lines were seeded and exposed to 10 μM Gltn for the indicated time periods. Cells were counted as described in A. Graphs represent n = />3 experiments carried out in triplicate triplicate +/- S.E. By six days post-treatment, all cell lines showed significant differences from control group. p = /< 0.05 as denoted by asterisks.
Figure 2.
Gltn exposure results in S phase arrest.
(A) MDA-MB-468 cells treated with Gltn for indicated times were synchronized in G2/M using double Thymidine/Nocodozole mediated arrest. Cell cycle progression was assessed after release using propidium iodide staining detected by fluorescence activated cell sorting (FACS). Results are representative of three independent experiments. (B) MDA-MB-468 cells were exposed to Gltn or vehicle for 72 hours prior to labeling with BrdU. Cells were collected at the indicated time periods post-BrdU labeling. BrdU incorporation was quantified using fluorescence activated cell sorting. Cells residing above the solid line are considered to reside in the S phase compartment. Results are representative of triplicate experiments.
Figure 3.
Gltn regulates the expression of proliferation related genes.
(A) MDA-MB-468 cells were treated with Gltn in triplicate for three or four days as indicated. RNA from treated cells or controls were isolated and gene expression analyzed by Nanostring technology. The results represent the mean fold-change of three independent drug treatments. (B) qPCR validation of genes regulated in MDA-MB-468 cells by Gltn. n = />2 experiments carried out in triplicate +/- S.D. (C) Western blotting of cyclin expression after Gltn treatment for the indicated time periods. UV light serves as a positive control for CcnD1 degradation.
Figure 4.
Chk2 is activated in response to Gltn.
(A) Western blotting of Check point kinases in response to Gltn. UV light serves as a positive control for Chk1 and Chk2 phospohorylation. Results are representative of triplicate experiments. (B) MDA-MB-468 cells were treated with Gltn for 72 hours prior to BrdU labeling. BrdU was quantified using FACs and results are representative of three independent experiments. (C) Immunofluorescent images of γH2A.X in Gltn treated cells. Cells were treated with Gltn daily for the indicated time periods and probed with an anti-Ser-129-H2A.X antibody for two hours. Doxorubicin treatment serves as a positive control for DNA damage. Results are representative of three independent experiments.
Figure 5.
Gltn effectively suppresses triple-negative tumor outgrowth in vivo.
(A) Mammary fat pads of athymic nude mice were injected with 1×106 triple-negative MDA-MB-468 cells. Subsequent to tumor palpation, mice were exposed to Gltn through drinking water or intraperitoneal injections. Tumor volume was measured using digital calipers +/- S.D. The results represent two independent mouse experiments with n = /> 4 mice per group in each experiment. “*” indicates difference between experimental and control group has p<0.05. (B) Morphology of Gltn exposed tumors shows diminished size and increased pallor at 30 days post treatment. (C) Weight of mice exposed to Gltn compared to control mice +/- S.D. (D) Immunohistochemistry of CcnD1 levels in residual tumors from mice exposed to Gltn orally. CcnD1 expression was shown to be diminished in these tumors.