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. 2018 Sep;57(9):1166-1180.
doi: 10.1002/mc.22833. Epub 2018 May 18.

Bitter Melon Juice Exerts Its Efficacy Against Pancreatic Cancer via Targeting Both Bulk and Cancer Stem Cells

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Free PMC article

Bitter Melon Juice Exerts Its Efficacy Against Pancreatic Cancer via Targeting Both Bulk and Cancer Stem Cells

Deepanshi Dhar et al. Mol Carcinog. .
Free PMC article

Abstract

Pancreatic cancer (PanC) is one of the deadliest malignancies worldwide and frontline treatment with gemcitabine becomes eventually ineffective due to increasing PanC resistance, suggesting additional approaches are needed to manage PanC. Recently, we have shown the efficacy of bitter melon juice (BMJ) against PanC cells, including those resistant to gemcitabine. As cancer stem cells (CSCs) are actively involved in PanC initiation, progression, relapse and drug-resistance, here we assessed BMJ ability in targeting pancreatic cancer-associated cancer stem cells (PanC-CSCs). We found BMJ efficacy against CD44+ /CD24+ /EpCAMhigh enriched PanC-CSCs in spheroid assays; BMJ also increased the sensitivity of gemcitabine-resistant PanC-CSCs. Exogenous addition of BMJ to PanC-CSC generated spheroids (not pre-exposed to BMJ) also significantly reduced spheroid number and size. Mechanistically, BMJ effects were associated with a decrease in the expression of genes and proteins involved in PanC-CSC renewal and proliferation. Specifically, immunofluorescence staining showed that BMJ decreases protein expression/nuclear localization of CSC-associated transcription factors SOX2, OCT4 and NANOG, and CSC marker CD44. Immunohistochemical analysis of MiaPaCa2 xenografts from BMJ treated animals also showed a significant decrease in the levels of CSC-associated transcription factors. Together, these results show BMJ potential in targeting PanC-CSC pool and associated regulatory pathways, suggesting the need for further investigation of its efficacy against PanC growth and progression including gemcitabine-resistant PanC.

Keywords: bitter melon juice; cancer stem cells; chemo-resistance; natural/dietary agents; pancreatic cancer.

Figures

Figure 1.
Figure 1.. BMJ affects both unsorted and CSC enriched PanC cell spheroids.
FACS sorting of untreated PANC1 cell line with CD44+/CD24+/EpCAM+ combination of CSC markers (A). The sorted CD44+/CD24+/EpCAM+ was subjected to limiting dilution assay to generate spheroids with cell number ranging from 200–10,000 cells per well for determining the stemness of triple positive population (B). The effect of BMJ was studied by exposing FACS sorted and unsorted cells to varying BMJ concentrations and recording the number of spheroids formed per well (C). p ≤ 0.05 is denoted by * and p ≤ 0.01 is denoted by ***.
Figure 2.
Figure 2.. BMJ treatment helps confer sensitivity to gemcitabine resistant PanC cells generated spheroids.
PANC1 and AsPC1 PanC cells surviving 2.5 μM gemcitabine treatment (A), and 5 μM gemcitabine treatment (B), were exposed to 0.5 or 2% BMJ (v/v) treatments in spheroid assay, and the decrease in spheroid number and size was recorded after 11days. Representative spheroid images are shown. p ≤ 0.05 is denoted by * and p ≤ 0.01 is denoted by ***.
Figure 3.
Figure 3.. BMJ affects the cell viability and stemness in PanC cell monolayers.
Trypan blue exclusion assay was performed for PanC cell lines MiaPaCa2, PANC1 and AsPC1 with 2–4% BMJ (v/v). The decrease in total cell number (A) and increase in percent cell death (B), are shown in the top panels for 24, 48 and 72 hours. Also, BMJ effect on PanC stemness was assessed by spheroid assays for PanC cell lines MiaPaCa2, PANC1 and AsPC1 where the cell monolayers were treated with 0.5–2% BMJ (v/v) single (C), and multiple treatments (D) and observed for 11 days. Representative spheroid images are shown. p ≤ 0.01 is denoted by ** and p ≤ 0.001 is denoted by ***.
Figure 4.
Figure 4.. BMJ affects number and size of spheres formed by PanC-CSCs.
PanC cells seeded for sphere formation were exposed to single and multiple treatments of BMJ (0.5–2% BMJ, v/v) and observed for 11 days (A). PanC cells were allowed to generate spheroids for 5 days and then treated with 2% BMJ to observe the effects on pre-formed spheroid number and size (B). Representative spheroid images are shown. p ≤ 0.05 is denoted by *, p ≤ 0.01 is denoted by ** and p ≤ 0.001 is denoted by ***.
Figure 5.
Figure 5.. BMJ alters the mRNA levels of PanC-CSC associated regulatory molecules and transcription factors.
MiaPaCa2 cells were exposed to 2% BMJ for 72 hours, thereafter harvested and subjected to RT2-qPCR array of human stem cell transcription factors for assessment of BMJ effects on gene expression of PanC-CSC associated transcription factors. Top panel depicts scatter plot showing BMJ vs untreated control group (1.5 fold level changes); downregulated genes are displayed as open circles (green). Bottom panel depicts genes displaying the most significant down-regulation with BMJ exposure.
Figure 6.
Figure 6.. BMJ decreases the protein expression of CSC associated transcription factors and surface markers in PanC cell monolayers.
Immunofluorescence staining of PanC cell monolayers treated with 2% BMJ for 72 hours shows a reduction in SOX2, OCT4, NANOG and CD44 protein expression levels in MiaPaCa2 (A), PANC1 (B), and AsPC1 (C) cells. The results from densitometry by Image J software are also provided and correlate to the data from the images. p ≤ 0.05 is denoted by *, p ≤ 0.01 is denoted by ** and p ≤ 0.001 is denoted by ***.
Figure 7.
Figure 7.. BMJ decreases the protein expression of CSC associated transcription factor SOX2 and CSC surface marker CD44 in PanC cell generated spheroids.
PanC cells were treated with 2% BMJ (72 hours) and allowed to generate spheroids in stem cell media for 10 days. On day 11, spheroids were harvested and stained by immunofluorescence for SOX2 and CD44 in MiaPaCa2 (A), PANC1 (B), and AsPC1 (C) spheroids. Z-stacking was performed for the spheroids. The results from densitometry by Image J software are also provided and correlate to the data from the images. p ≤ 0.05 is denoted by * and p ≤ 0.01 is denoted by **.
Figure 8.
Figure 8.. BMJ targets and downregulates the expression of CSC associated transcription factors in MiaPaCa2 xenografts.
Paraffin embedded MiaPaCa2 xenograft tumor sections from control and BMJ treated groups were subjected to immunohistochemical analysis for determining the in vivo effect on the protein levels of PanC-CSC associated transcription factors: SOX2 (A), OCT4 (B), NANOG (C). and PDX1 (D). The percent positive cells were quantitated by counting the brown stained nuclei for each molecule in control versus the BMJ treated groups. p ≤ 0.05 is denoted by * and p ≤ 0.001 is denoted by ***.

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