Phytochemical Inhibition of Multidrug Resistance Protein-1 as a Therapeutic Strategy for Hemangioendothelioma

Abstract

Aims: Hemangiomas are endothelial cell tumors and the most common soft tissue tumors in infants. They frequently cause deformity and can cause death. Current pharmacologic therapies have high-risk side-effect profiles, which limit the number of children who receive treatment. The objectives of this work were to identify the mechanisms through which standardized berry extracts can inhibit endothelial cell tumor growth and test these findings in vivo.

Results: EOMA cells are a validated model that generates endothelial cell tumors when injected subcutaneously into syngeneic (129P/3) mice. EOMA cells treated with a blend of powdered natural berry extracts (NBE) significantly inhibited activity of multidrug resistance protein-1 (MRP-1) compared to vehicle controls. This resulted in nuclear accumulation of oxidized glutathione (GSSG) and apoptotic EOMA cell death. When NBE-treated EOMA cells were injected into mice, they generated smaller tumors and had a higher incidence of apoptotic cell death compared to vehicle-treated EOMA cells as demonstrated by immunocytochemistry. Kaplan-Meier survival curves for tumor-bearing mice showed that NBE treatment significantly prolonged survival compared to vehicle-treated controls.

Innovation: These are the first reported results to show that berry extracts can inhibit MRP-1 function that causes apoptotic tumor cell death by accumulation of GSSG in the nucleus of EOMA cells where NADPH oxidase is hyperactive and causes pathological angiogenesis.

Conclusions: These findings indicate that berry extract inhibition of MRP-1 merits consideration and further investigation as a therapeutic intervention and may have application for other cancers with elevated MRP-1 activity. Antioxid. Redox Signal. 26, 1009-1019.

Keywords: apoptosis; glutathione; hemangioendothelioma; multidrug resistance protein 1.

Conflict of interest statement

Chandan K. Sen is a shareholder in NutrimiR LLC, which supplied the NBE product. For all other authors, no competing financial interests exist.

Figures

FIG. 1.

NBE inhibits MRP-1 activity. (A) LDH toxicity assay of EOMA cells treated with NBE shows a dose-dependent decrease in EOMA cell survival. MAE cells were included as nontumor-forming endothelial cell controls. (B) MRP-1 activity was measured by flow cytometry detection of calcein exclusion and was found to be significantly reduced in EOMA cells treated with NBE at 50 and 100 μg/ml and in MAE cells treated with 100 μg/ml dose. (C, D) mRNA and Western blot data show reduced MRP-1 protein expression in EOMA treated with NBE (50 μg/ml) compared to vehicle control. *p < 0.05, n = 6. LDH, lactate dehydrogenase; MAE, murine aortic endothelial; MFU, mean fluorescence units; MRP-1, multidrug resistance protein-1; NBE, natural berry extracts.

FIG. 2.

NBE decreases nuclear MRP-1 localization. (A) Immunofluorescence detection of MRP-1 (green) in NBE-treated EOMA cells labeled with DAPI (blue) shows significant reduction in green fluorescence compared to vehicle treatment (scale bar = 50 μm). Pixel densitometry was used to quantitate fluorescence intensity (AxioVision Rel 4.8 software; Zeiss). (B) Nuclear colocalization of green and blue fluorescence as shown by white dots reveals a significant loss of nuclear MRP-1 in response to NBE treatment (scale bar = 50 μm). (C) MRP-1 intensity ratio of nuclear to cytosolic compartment (total-nuclear) in vehicle control and NBE treatment groups. *p < 0.05, n = 4. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

NBE-mediated loss of MRP-1 results in nuclear accumulation of GSSG. GSH and GSSG content in whole cell lysates and cytosolic and nuclear fractions of NBE-treated (50 μg/ml) EOMA cells were measured using a commercially available kit according to the manufacturer's instructions. (A) GSSG measurements, (B) GSH measurements, and (C) GSSG/GSH ratio in whole cell lysates of vehicle- and NBE-treated EOMA cells. BCNU (10 μM, 24 h) is an inhibitor of glutathione reductase and was used as a positive control for GSSG accumulation. (D) Cell viability was measured by flow cytometry detection of PI for all treatment groups. (E) Nuclear and cytosolic fraction measurement of GSSG, (F) GSH, and (G) GSSG/GSH ratio shows significant accumulation of nuclear GSSG in the NBE-treated group compared to control with a corresponding increase in GSSG/GSH ratio. (H) Comparison of nuclear versus cytosolic GSSG/GSH ratio *p < 0.05, n = 4. BCNU, 1,3-bis (2-chloroethyl)-N-nitrosourea; GSH, reduced glutathione; GSSG, oxidized glutathione; PI, propidium iodide.

FIG. 4.

NBE treatment causes apoptotic cell death. (A) NBE-induced EOMA cell death is predominantly apoptotic as measured by the Apoptosis/Necrosis Assay Kit where Apopxin Green (apoptosis marker—green), 7-AAD (necrosis marker—red), and Cytocalcein Violet (cell viability marker—blue) were used (scale bar = 50 μm). (B) Pixel densitometry was used to quantify fluorescence signals and showed significantly elevated incidence of apoptosis compared to necrosis after NBE treatment. (C, D) Western blot detection of apoptotic proteins dimeric/activated Bax, caspase 9, and cleaved caspase 3 was significantly elevated in NBE-treated EOMA cells compared to vehicle-treated controls. (E, F) Protein expression results were confirmed by immunocytochemistry on EOMA cells treated with NBE or vehicle control with fluorescence intensity quantified using pixel densitometry (scale bar = 25 μm). The results are consistent with Western blot findings. *p < 0.05, n = 6. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

NBE treatment inhibits tumor growthin vivo. Syngeneic 6–8-week-old female 129 P/3 mice received a subcutaneous injection of EOMA cells treated with NBE (n = 6) or vehicle control (n = 6) for 12 days before injection. (A) Photos depict representative tumor length, width, and projection 5 and 10 days after EOMA cell injection. (B) Tumor volume was quantified using calipers (length × width × height). Significantly reduced tumor growth was observed at day 10 in NBE-treated group compared to vehicle control. (C) Kaplan–Meier survival curve analyzed by log-rank analysis shows that mice injected with NBE-treated EOMA cells have a significant survival advantage compared to vehicle control. *p < 0.05. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

NBE specimens have fewer tumor-forming EOMA cells. (A) Serial sections of whole tumor specimens 10 days after injection of EOMA cells treated with vehicle control or NBE. There were two tissue sections per group with pixel densitometry done on four separate areas per section using a 20 × magnification objective with a 5 × zoom for each area of analysis. (i) H&E staining of vehicle control or NBE (iv). EOMA cells within the tumor stroma were identified by Lyve-1 immunohistochemistry (1:100) performed on vehicle control (ii) and NBE (v) group with negative control (secondary antibody alone) of vehicle control (iii) and NBE (vi) group. (B) Quantification of Lyve-1 expression (pixel densitometry) showed significantly decreased levels of Lyve-1 protein in NBE-treated compared to vehicle control group. Scale bar = 200 μM. Results are expressed as mean ± standard deviation of at least three determinant sections; *p < 0.05 (n = 3). H&E, hematoxylin and eosin. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

NBE treatment induces endothelial tumor apoptosisin vivo.(A) There were two tissue sections per group with pixel densitometry done on four separate areas per section using a 20 × magnification objective with a 5 × zoom for each area of analysis. Immunofluorescence detection of cleaved Bax (1:100), caspase 9 (1:100), and cleaved caspase 3 (1:200) proteins in mouse tumor tissue sections collected 10 days after EOMA cell injection. DAPI was used to counterstain the nucleus (scale bar = 100 μm, inset scale bar = 25 μm). Fluorescence intensity analysis showed significant differences in all apoptosis marker proteins in NBE-treated group compared to vehicle treatment. (B) Western blot shows that NBE-treated tumors have significantly lower levels of MRP-1 than vehicle controls *p < 0.05, n = 6. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars