Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jan;51(1):7.
doi: 10.3892/or.2023.8666. Epub 2023 Nov 17.

Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

Ji He  1 , Piyush Bugde  1 , Jiawei Li  1 , Riya Biswas  1 , Siting Li  1 , Xuewei Yang  2 , Fang Tian  3 , Zimei Wu  4 , Yan Li  1
Affiliations

Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

Ji He et al. Oncol Rep. 2024 Jan.

Abstract

Gemcitabine‑based chemotherapy has been widely adopted as the standard and preferred chemotherapy regimen for treating advanced pancreatic cancer. However, the contribution of multidrug resistance protein 5 (MRP5) to gemcitabine resistance and pancreatic cancer progression remains controversial. In the present study, the effect of silencing MRP5 on gemcitabine resistance and cell proliferation and migration of human pancreatic cancer MIA Paca‑2 and PANC‑1 cells was investigated by using short‑hairpin RNA delivered by lentiviral vector transduction. The knockdown of MRP5 was confirmed on both mRNA and protein levels using qPCR and surface staining assays, respectively. MRP5‑regulated gemcitabine sensitivity was assessed by MTT, PrestoBlue and apoptosis assays. The effect of MRP5 on pancreatic cancer cell proliferation and migration was determined using colony‑formation, wound‑healing and Transwell migration assays. The interaction of gemcitabine and cyclic guanosine monophosphate (cGMP) with MRP5 protein was explored using molecular docking. The results indicated that the MRP5 mRNA and protein levels were significantly reduced in all the MIA Paca‑2 and PANC‑1 clones. MRP5 affected gemcitabine cytotoxicity and the rate of gemcitabine‑induced apoptosis. Silencing MRP5 decreased cell proliferation and migration in both MIA Paca‑2 and PANC‑1 cells. Docking studies showed high binding affinity of cGMP towards MRP5, indicating the potential of MRP5‑mediated cGMP accumulation in the microenvironment. In conclusion, MRP5 has an important role in cancer proliferation and migration in addition to its drug efflux functions in two widely available pancreatic tumour cell lines (MIA Paca‑2 and PANC‑1).

Keywords: ABC transporters; MRP5; gemcitabine; multidrug resistance; pancreatic cancer.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Functional overexpression of MRP5 in human pancreatic cancer MIA Paca-2 and PANC-1 cells. (A) High MRP5 mRNA expression in both MIA PaCa-2 and PANC-1 cells (in red colour) compared to other pancreatic cancer cell lines from the Wagner dataset stored in ONCOMINE (https://www.oncomine.org). (B and C) MRP5 protein detected in representative flow cytometry histogram of cell surface staining using the anti-MRP5 primary antibody (red) and isotype control IgG2a (green) on (B) MIA Paca-2 and (C) PANC-1 cells. Both the primary antibody and isotype control were labelled with Alexa Fluor 488 secondary antibody. The x-axis is the fluorescence signal intensity displayed in a linear log scale. (D) Functional expression of MRP5 detected by BCECF accumulation in MIA Paca-2 and PANC-1 cells at 15 min in the presence and absence of 50 µM benzbromarone. All data are normalised to the fluorescence intensity determined in the absence of benzbromarone. The bars represent the mean and standard deviation from three independent experiments performed in triplicates. ****P<0.0001 according to Sidak's post-hoc test that followed two-way ANOVA. MRP5, multidrug resistance protein 5.
Figure 2.
Figure 2.
MRP5 expression at the mRNA level in (A) MIA Paca-2 and (B) PANC-1 clones transduced with scrambled control and multidrug resistance protein 5-short-hairpin RNA. Relative MRP5 mRNA expression was detected by reverse transcription-quantitative PCR. MRP5 mRNA expression was normalised to the reference gene GAPDH and relative quantitation of gene expression was calculated using the comparative threshold cycle method (2−ΔΔCq). All data were expressed as the mean and standard deviation from three independent experiments performed in duplicates. Cell surface protein expression of MRP5 in (C) MIA Paca-2 and (D) PANC-1 cells presented as the mean percentage of the scrambled control. The bar represents the mean and standard deviation from three independent experiments performed in triplicates. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 from Dunnett's post-hoc test that followed one-way ANOVA for comparison of all MRP5 knockdown clones to the scrambled control. (E and F) MRP5 protein detected in representative flow cytometry histograms of cell surface staining using anti-MRP5 primary antibody and Alexa Fluor 488 secondary antibody on (E) MIA Paca-2 and (F) PANC-1 clones. The x-axis is the fluorescence signal intensity displayed in a linear log scale. MRP5, multidrug resistance protein 5; shRNA, short-hairpin RNA.
Figure 3.
Figure 3.
Representative gemcitabine-induced inhibition of growth of MIA Paca-2 and PANC-1 clones transduced with scrambled control and multidrug resistance protein 5-short-hairpin RNA in 2D and 3D culture models. (A) IC50 values were decreased by 52% (M c1, P=0.0002) and 42% (M c2, P=0.0015) in 2D culture model of MIA Paca-2 cells; (B) IC50 values were decreased by 46% (P c1, P=0.0006) and 41% (P c2, P=0.0013) 2D model for PANC-1; (C) 3D model for MIA Paca-2; and (D) 3D model for PANC-1. Values are expressed as the mean and standard deviation (n=3). Solid lines are non-linear regression fits (Y=Bottom+(Top-Bottom)/[1+10(LogIC50-X)] to the data. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 according to Sidak's post-hoc tests that followed two-way ANOVA. Con., concentration.
Figure 4.
Figure 4.
Representative gemcitabine-induced apoptosis in MIA PaCa-2 (A and B) and PANC-1 (C and D) clones transduced with scramble control and multidrug resistance protein 5-short-hairpin RNA. MIA Paca-2 and PANC-1 clones were treated with gemcitabine (100 and 200 nM) for 24 and 48 h, respectively. The apoptotic cells were stained with CellEvent™ Caspase-3/7 Green Detection Reagent and visualised by fluorescence microscopy (scale bars, 100 µm). The fluorescence intensity was then measured using a plate reader. Data are presented as a mean percentage of vehicle control. The bars represent the mean and standard deviation from three independent experiments performed in triplicates. *P<0.05; **P<0.01; ***P<0.001 according to Sidak's post-hoc test that followed two-way ANOVA. Con., concentration.
Figure 5.
Figure 5.
Representative clonogenic growth images of (A) MIA Paca-2 and (B) PANC-1 clones transduced with scrambled control and multidrug resistance protein 5-short-hairpin RNA. MIA Paca-2 and PANC-1 clones at a density of 200 cells/well were seeded into a 6-well plate and incubated for 10 days. Colonies of >50 cells were scored for survival and counted using ImageJ 1.52g software. The number of colonies formed by (C) MIA Paca-2 and (D) PANC-1 cells is presented as a mean percentage of the scrambled control. The bars represent the mean and standard deviation from three independent experiments performed in duplicates. *P<0.05; ***P<0.001; ****P<0.0001 from Dunnett's post-hoc test that followed one-way ANOVA.
Figure 6.
Figure 6.
Representative images for the wound-healing assay using (A) MIA Paca-2 and (B) PANC-1 clones transduced with scrambled control and multidrug resistance protein 5-short-hairpin RNA. Quantified scratch wound coverage in (C) MIA Paca-2 and (D) PANC-1 cells. Images taken at the designated time-points (total magnification, ×40) were analysed by ImageJ 1.52g software and data are presented as a mean percentage of the control (0 h). The bars represent the mean and standard deviation from three independent experiments performed in triplicates. *P<0.05; **P<0.01; ****P<0.0001 from Dunnett's post-hoc test that followed two-way ANOVA.
Figure 7.
Figure 7.
Representative Transwell migration images of (A) MIA Paca-2 and (B) PANC-1 clones transduced with scrambled control and multidrug resistance protein 5-short-hairpin RNA (scale bars, 200 µm). Migrated cells were quantified as a mean percentage of scrambled control in (C) MIA Paca-2 and (D) PANC-1 cells. The bars represent the mean and standard deviation from three independent experiments performed in duplicates. ***P<0.001; ****P<0.0001 from Dunnett's post-hoc test that followed one-way ANOVA.
Figure 8.
Figure 8.
Ligands docked on MRP5 macromolecule. Binding of (A) gemcitabine and (B) cyclic guanosine monophosphate with MRP5. MRP5 is depicted in red colour in ribbon form. MRP5, multidrug resistance protein 5.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Adamska A, Domenichini A, Falasca M. Pancreatic ductal adenocarcinoma: Current and evolving therapies. Int J Mol Sci. 2017;18:1338. doi: 10.3390/ijms18071338. - DOI - PMC - PubMed
    1. Von Hoff DD, Ervin T, Arena FP, Chiorean EG, Infante J, Moore M, Seay T, Tjulandin SA, Ma WW, Saleh MN, et al. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N Engl J Med. 2013;369:1691–1703. doi: 10.1056/NEJMoa1304369. - DOI - PMC - PubMed
    1. Conroy T, Desseigne F, Ychou M, Bouché O, Guimbaud R, Bécouarn Y, Adenis A, Raoul JL, Gourgou-Bourgade S, de la Fouchardière C, et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med. 2011;364:1817–1825. doi: 10.1056/NEJMoa1011923. - DOI - PubMed
    1. Adamska A, Ferro R, Lattanzio R, Capone E, Domenichini A, Damiani V, Chiorino G, Akkaya BG, Linton KJ, De Laurenzi V, et al. ABCC3 is a novel target for the treatment of pancreatic cancer. Adv Biol Regul. 2019;73:100634. doi: 10.1016/j.jbior.2019.04.004. - DOI - PubMed
    1. Gnanamony M, Gondi CS. Chemoresistance in pancreatic cancer: Emerging concepts. Oncol Lett. 2017;13:2507–2513. doi: 10.3892/ol.2017.5777. - DOI - PMC - PubMed

Grants and funding

This research was funded by the Faculty of Health and Environmental Science (Auckland University of Technology, NZ) Research and Development Fund 2022.
-