pubmed.ncbi.nlm.nih.gov

Antiproliferative Activity of Krukovine by Regulating Transmembrane Protein 139 (TMEM139) in Oxaliplatin-Resistant Pancreatic Cancer Cells - PubMed

  • ️Sun Jan 01 2023

Antiproliferative Activity of Krukovine by Regulating Transmembrane Protein 139 (TMEM139) in Oxaliplatin-Resistant Pancreatic Cancer Cells

Jee-Hyung Lee et al. Cancers (Basel). 2023.

Abstract

Krukovine (KV) is an alkaloid isolated from the bark of Abuta grandifolia (Mart.) Sandw. (Menispermaceae) with anticancer potential in some cancers with KRAS mutations. In this study, we explored the anticancer efficacy and mechanism of KV in oxaliplatin-resistant pancreatic cancer cells and patient-derived pancreatic cancer organoids (PDPCOs) with KRAS mutation. After treatment with KV, mRNA and protein levels were determined by RNA-seq and Western blotting, respectively. Cell proliferation, migration, and invasion were measured by MTT, scratch wound healing assay, and transwell analysis, respectively. Patient-derived pancreatic cancer organoids (PDPCOs) with KRAS mutations were treated with KV, oxaliplatin (OXA), and a combination of KV and OXA. KV suppresses tumor progression via the downregulation of the Erk-RPS6K-TMEM139 and PI3K-Akt-mTOR pathways in oxaliplatin-resistant AsPC-1 cells. Furthermore, KV showed an antiproliferative effect in PDPCOs, and the combination of OXA and KV inhibited PDPCO growth more effectively than either drug alone.

Keywords: KRAS; combination; krukovine; metastasis; oxaliplatin; pancreatic cancer; patient-derived pancreatic cancer organoid (PDPCO); transmembrane protein 139 (TMEM139).

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

(A) The structure of krukovine. (B) Cell viability of KV-treated pancreatic cancer cell lines. The viability of pancreatic cancer cells was measured using the MTT assay following treatment with the indicated concentrations of KV for 48 h. All data are presented as mean ± SD (n = 3, * p < 0.05, *** p < 0.001). (C) Oxaliplatin-resistant and non-oxaliplatin-resistant AsPC-1 cells; microscopic images. Scale bar: 50 μm. Microscopic images of AsPC-1 cells were measured using the light microscope following treatment with the indicated concentration of oxaliplatin (100 μM) for 48 h. (D) Cell viability of oxaliplatin-resistant AsPC-1 cells treated with oxaliplatin and KV. The viability of oxaliplatin-resistant AsPC-1 cells was measured using the MTT assay following treatment with the indicated concentrations of oxaliplatin or KV for 48 h. Data are presented as the mean ± SD (n = 3, * p < 0.05, *** p < 0.001).

Figure 1
Figure 1

(A) The structure of krukovine. (B) Cell viability of KV-treated pancreatic cancer cell lines. The viability of pancreatic cancer cells was measured using the MTT assay following treatment with the indicated concentrations of KV for 48 h. All data are presented as mean ± SD (n = 3, * p < 0.05, *** p < 0.001). (C) Oxaliplatin-resistant and non-oxaliplatin-resistant AsPC-1 cells; microscopic images. Scale bar: 50 μm. Microscopic images of AsPC-1 cells were measured using the light microscope following treatment with the indicated concentration of oxaliplatin (100 μM) for 48 h. (D) Cell viability of oxaliplatin-resistant AsPC-1 cells treated with oxaliplatin and KV. The viability of oxaliplatin-resistant AsPC-1 cells was measured using the MTT assay following treatment with the indicated concentrations of oxaliplatin or KV for 48 h. Data are presented as the mean ± SD (n = 3, * p < 0.05, *** p < 0.001).

Figure 2
Figure 2

The Kaplan–Meier survival curve according to the TMEM139 expression level. The Kaplan–Meier survival curve represents the overall survival (OS) and relapse-free survival (RFS) of pancreatic cancer patients according to the TMEM139 expression level.

Figure 3
Figure 3

(A) The effect of KV on TMEM139 protein, the TMEM139-associated signaling pathway, and the PI3K-Akt-mTOR signaling pathway in oxaliplatin-resistant AsPC-1 cells. Oxaliplatin-resistant AsPC-1 cells were treated with the indicated concentrations of KV for 48 h, and the protein expression level of TMEM139 protein, the TMEM139-associated signaling pathway, and the PI3K-Akt-mTOR signaling pathway was determined by Western blotting. α-tubulin was used as an internal control. (B) Protein–protein interaction (PPI) for TMEM139 from the search tool (STRING) database. Interacting proteins for TMEM139 (Transmembrane 139) Gene: MAPK3/1 (Mitogen-Activated Protein Kinase 3/1; Erk1/2)-RPS6KA3 (Ribosomal Protein S6 Kinase A3)-TMEM139 (C) Protein–protein interaction (PPI) for PI3K-Akt and PI3K-Akt-mTOR signaling pathway from the search tool (STRING) database. Interacting proteins for Akt1 (AKT Serine/Threonine Kinase) gene: PI3K-Akt and PI3K-Akt-mTOR.

Figure 3
Figure 3

(A) The effect of KV on TMEM139 protein, the TMEM139-associated signaling pathway, and the PI3K-Akt-mTOR signaling pathway in oxaliplatin-resistant AsPC-1 cells. Oxaliplatin-resistant AsPC-1 cells were treated with the indicated concentrations of KV for 48 h, and the protein expression level of TMEM139 protein, the TMEM139-associated signaling pathway, and the PI3K-Akt-mTOR signaling pathway was determined by Western blotting. α-tubulin was used as an internal control. (B) Protein–protein interaction (PPI) for TMEM139 from the search tool (STRING) database. Interacting proteins for TMEM139 (Transmembrane 139) Gene: MAPK3/1 (Mitogen-Activated Protein Kinase 3/1; Erk1/2)-RPS6KA3 (Ribosomal Protein S6 Kinase A3)-TMEM139 (C) Protein–protein interaction (PPI) for PI3K-Akt and PI3K-Akt-mTOR signaling pathway from the search tool (STRING) database. Interacting proteins for Akt1 (AKT Serine/Threonine Kinase) gene: PI3K-Akt and PI3K-Akt-mTOR.

Figure 4
Figure 4

(A) The effect of KV on oxaliplatin-resistant AsPC-1 cells’ migration. Monolayers of oxaliplatin-resistant AsPC-1 cells were scratched mechanically and treated with KV (indicated concentrations) for 48 h. Representative images of wound closure obtained under a light microscope. All data are presented as mean ± SD (n = 3, *** p < 0.001). (B) The effect of KV on oxaliplatin-resistant AsPC-1 cell invasion. Oxaliplatin-resistant AsPC-1 cells were pretreated with indicated concentrations of KV for 24 h, reseeded into the upper chamber of transwell inserts, and incubated for 24 h. The cells that invaded lower chambers were then fixed, stained, imaged, and counted. All data are presented as mean ± SD (n = 3, *** p < 0.001). (C) Top 20 terms of GO (Gene Ontology) functional analysis. RNA-seq assay was performed in oxaliplatin-resistant AsPC-1 cells treated with KV (25 μM) and compared with a control (PBS with DMSO-treated). The dot color represents the p-values. The scale of the spots indicates the number of genes involved.

Figure 4
Figure 4

(A) The effect of KV on oxaliplatin-resistant AsPC-1 cells’ migration. Monolayers of oxaliplatin-resistant AsPC-1 cells were scratched mechanically and treated with KV (indicated concentrations) for 48 h. Representative images of wound closure obtained under a light microscope. All data are presented as mean ± SD (n = 3, *** p < 0.001). (B) The effect of KV on oxaliplatin-resistant AsPC-1 cell invasion. Oxaliplatin-resistant AsPC-1 cells were pretreated with indicated concentrations of KV for 24 h, reseeded into the upper chamber of transwell inserts, and incubated for 24 h. The cells that invaded lower chambers were then fixed, stained, imaged, and counted. All data are presented as mean ± SD (n = 3, *** p < 0.001). (C) Top 20 terms of GO (Gene Ontology) functional analysis. RNA-seq assay was performed in oxaliplatin-resistant AsPC-1 cells treated with KV (25 μM) and compared with a control (PBS with DMSO-treated). The dot color represents the p-values. The scale of the spots indicates the number of genes involved.

Figure 5
Figure 5

(A) Mutation profile of PDPCOs. The main representative mutations (KRAS and TP53) in PDPCOs (patient-derived pancreatic cancer organoids) were analyzed through whole-exome sequencing analysis. (B) Antiproliferative effect of KV on PDPCOs. PDPCOs (SNU-4206, SNU-4305-TO, SNU-4425-TO, SNU-3947-TO, and SNU-4340-TO) were treated with various concentrations of KV and detected by 3D cell titer glow assay after 72 h. All data are presented as mean ± SD (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001). (C) KV inhibited the growth of the tested PDPCO (SNU-4340-TO) in a dose-dependent manner. Representative images of the organoid treated with KV obtained under a light microscope. (D) Cell viability assay for measuring the combination effect of KV with oxaliplatin. The multidrug-resistant organoid (SNU-4425-TO) was treated with KV and/or oxaliplatin for 72 h with indicated concentration (con; control, K 11.1; krukovine 11.1 μM, O 33.3; oxaliplatin 33.3 μM, K 11.1 + O 33.3; krukovine 11.1 μM + oxaliplatin 33.3 μM) and detected by 3D cell titer glow assay. All data are presented as mean ± SD (n = 3). All data are presented as mean ± SD (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001). (E) Combination effect of KV with oxaliplatin on the growth of multidrug-resistant PDPCO (SNU-4425-TO). Representative images of the organoid treated with KV and/ or oxaliplatin for 72 h with the indicated concentrations (CON; control, K 11.1; krukovine 11.1 μM, O 33.3; oxaliplatin 33.3 μM, K 11.1 + O 33.3; krukovine 11.1 μM + oxaliplatin 33.3 μM). Images were obtained under a light microscope. (F) Effects of KV on the cleaved-PARP expression in multidrug-resistant organoid (SNU-4425-TO). SNU-4425-TO were treated with the indicated concentrations of KV for 72 h, and the protein expression level was determined by Western blotting. α-tubulin was used as an internal control.

Similar articles

Cited by

References

    1. Iovanna J., Mallmann M.C., Gonçalves A., Turrini O., Dagorn J.-C. Current knowledge on pancreatic cancer. Front. Oncol. 2012;2:6. doi: 10.3389/fonc.2012.00006. - DOI - PMC - PubMed
    1. Miller K.D., Nogueira L., Mariotto A.B., Rowland J.H., Yabroff K.R., Alfano C.M., Ahmedin Jemal D.V.M., Kramer J.L., Siege R.L. Cancer treatment and survivorship statistics, 2019. CA Cancer J. Clin. 2019;69:363–385. doi: 10.3322/caac.21565. - DOI - PubMed
    1. Rawla P., Sunkara T., Gaduputi V. Epidemiology of pancreatic cancer: Global trends, etiology and risk factors. World J. Oncol. 2019;10:10–27. doi: 10.14740/wjon1166. - DOI - PMC - PubMed
    1. Li Q., Feng Z., Miao R., Liu X., Liu C., Liu Z. Prognosis and survival analysis of patients with pancreatic cancer: Retrospective experience of a single institution. World J. Surg. Oncol. 2022;20:11. doi: 10.1186/s12957-021-02478-x. - DOI - PMC - PubMed
    1. Otsu T., Inokawa Y., Takami H., Hayashi M., Kurimoto K., Tanaka N., Shimizu D., Hattori N., Kanda M., Tanaka C., et al. Comparison between FOLFIRINOX and nal-IRI/FL as Second-line Treatment After Gemcitabine Plus Nab-paclitaxel for Pancreatic Cancer. Anticancer Res. 2022;42:3889–3894. doi: 10.21873/anticanres.15882. - DOI - PubMed

Grants and funding

This research was supported by Chong Kun Dang pharmaceutical Corp. and by the Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (0720213063).

LinkOut - more resources