Abstract
Background/Aim: Methotrexate (MTX) resistance in osteosarcoma results in a very poor patient prognosis. We previously reported that super MTX-resistant osteosarcoma (143B-MTXSR) cells, selected from parental 143B osteosarcoma (143B-P) cells by culturing them with increasing concentrations of MTX, exhibited reduced malignancy, despite the over-expression of oncogenes. The present study explored the mechanism of reduced malignancy in the super MTX-resistant osteosarcoma cells. Materials and Methods: Previously selected 143B-MTXSR cells which are 5,500 times more MTX resistant than parental cells, were used for this study. The status of methylated histone H3K9me3 and H3K27me3 marks was examined with western immunoblotting and compared between 143B-MTXSR and parental 143B-P cells. Results: Histone H3K9me3 and H3K27me3 marks were over-expressed in 143B-MTXSR compared to 143B-P (p<0.05, p<0.01, respectively). Conclusion: Over-expression of histone H3K9me3 and H3K27me3 marks may be related to super-MTX resistance and to the loss of malignancy of super MTX-resistant osteosarcoma cells due to the fundamental relationship of methylation and cancer.
In osteosarcoma, the 5-year survival rate for patients who acquire resistance to first line-chemotherapy, such as doxorubicin (DOX), cisplatinum (CDDP), and high-dose methotrexate (MTX), is approximately 20%, which has not improved in 30 years (1, 2). In contrast, patients with osteosarcoma who respond to first-line chemotherapy have a 60-70% 5-year survival rate (3-5). Therefore, in order to improve treatment outcomes in osteosarcoma, further understanding of the mechanism of drug resistance is needed.
We previously established human 143B osteosarcoma cells which are 5,500-fold more MTX resistant than their parental cells, termed super-MTX-resistant osteosarcoma (143B-MTXSR) (6). The 143B-MTXSR cells over-express dihydrofolate reductase (DHFR), phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), and myelocytomatosis oncogene (c-MYC), but paradoxically lost malignancy.
It has been reported that methylated histone H3K9me3 and H3K27me3 marks repress gene expression, while methylated histone H3K4me3, H3K36me3, and H3K79me3 promote gene expression (7-12). Although there have been reports that histone-H3 methylated lysine marks, including H3K9me3 and H3K27me3, are related to drug resistance in various types of cancer, they are inconsistent.
In the present report, the super MTX-resistant osteosarcoma subline and parental cells were compared regarding the status of histone marks H3K9me3 and H3K27me3, to determine whether their changes could be correlated to the MTX-resistance and reduced malignancy of 143B-MTXSR cells.
Materials and Methods
Cell culture. The 143B human osteosarcoma cell line used in the present study was obtained from the American Type Culture Collection (#CRL-8303, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning Inc., Corning, NY, USA), which was supplemented with 10% fetal bovine serum (FBS) and 1 IU/ml penicillin/streptomycin.
Establishment of super-methotrexate-resistant osteosarcoma cells. The super-methotrexate-resistant 143B osteosarcoma cells (143B-MTXSR) were previously selected as reported (6). Briefly, parental 143B osteosarcoma cells (143B-P) were cultured with increasing concentrations of MTX (#HY-14519, MedChem Express, Monmouth Junction, NJ, USA) (0.04 μM to 100 μM) for 12 months.
Western immunoblotting. Extraction of histone protein from 143B-MTXSR and 143B-P cells and immunoblotting was performed as previously reported (13-19). Anti-H3K9me3 (1:1,000, #13969, Cell Signaling Technology, Danvers, MA, USA); anti-H3K27me3 (1:1,000, #9733, Cell Signaling Technology); anti-histone-H3 (1:1,500, 17168-1-AP, Proteintech, Rosemont, IL, USA) antibodies were used. The total-histone H3 antibody (1:1,500#17168-1-AP, Proteintech) was used as an internal loading control. Horseradish-peroxidase-conjugated anti-rabbit IgG (1:5,000, #SA00001-2, Proteintech) was used as the secondary antibody. The immunoblotting signals were detected with a UVP ChemStudio instrument (Analytik Jena, Upland, CA, USA), visualized with Clarity Western ECL Substrate (#1705060, Bio-Rad Laboratories, Hercules, CA, USA). Experiments were performed three times.
Statistical analysis. The Welch’s t-test was performed to compare two groups with Microsoft Excel for Mac ver. 16.86 (Microsoft, Redmond, WA, USA). Quantification of the histone-protein and histone-mark expression was calculated using ImageJ ver. 1.53k (National Institutes of Health, Bethesda, MD, USA). Bar graphs express the mean and error bars express standard deviation of the mean. A probability value of p≤0.05 was defined as a statistically-significant difference.
Results
Histone H3K9me3 and H9K27me3 marks in super-MTX-resistant osteosarcoma cells are over-expressed compared to parental osteosarcoma cells. Western immunoblotting was performed to compare the expression of H3K9me3 and H3K27me3 histone marks in 143B-MTXSR and parental 143B-P cells. 143B-MTXSR cells had increased expression of H3K9me3 by 2-fold (p<0.05) and H3K27me3 by 2.5-fold (p<0.01), compared to parental 143B-P cells (Figure 1).
Expression and quantification of histone H3K9me3 and H3K27me3 marks in methotrexate-resistant and parental 143B osteosarcoma cells. (A) Western immunoblotting for histone marks H3K9me3 and H3K27me3 from super-methotrexate-resistant 143B osteosarcoma (143B-MTXSR) cells and parental 143B osteosarcoma cells (143B-P). (B) Quantitation of histone marks H3K9me3 and H3K27me3 in 143B-MTXSR and parental 143B-P cells. *p<0.05, **p<0.01.
Discussion
Histone marks H3K9me3 and H3K27me3 have been reported to be related to drug resistance in various types of cancer. The expression level of histone mark H3K9me3 has shown both positive (20-30) and negative (31-36) correlation to drug resistance. The expression level of histone mark H3K27me3 has also shown both positive (22, 30, 37-62) and negative (21, 24, 31, 63-76) correlation to drug resistance.
The expression level of histone marks H3K9me3 (34) and H3K27me3 (66) showed a negative correlation with cisplatinum resistance. The correlation of the expression level of histone marks H3K9me3/H3K27me3 with the resistance to other first line chemotherapies, such as MTX, has not been previously reported. In the present study, the expression of histone marks H3K9me3 and H3K27me3, which are involved in gene repression, was increased in super MTX-resistant osteosarcoma 143B-MTXSR cells, compared to the parental 143B osteosarcoma cells.
Which histone-H3 lysine-methylation-modifications are directly involved in specific gene regulation is still incompletely understood (77, 78). However, resistance to MTX and reduction of malignancy in 143B-MTXSR cells may be attributed to epigenetic regulation of particular gene-expression changes via histone-marks H3K9me3 and H3K27me3 expression. Further research on the mechanism how methylated-histone-mark expression affects drug resistance and malignancy is necessary, especially since altered methylation is the fundamental hallmark of cancer (13-19, 79-95).
Acknowledgements
This article is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Professor Gordon H. Sato, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Jack Geller, MD, Shigeo Yagi, PhD, J.A.R. Mead, PhD, Eugene P. Frenkel, MD, Professor Sheldon Penman, Professor John R. Raper, John Mendelsohn, MD and Joseph Leighton, MD.
Footnotes
Authors’ Contributions
YA and RMH were involved in study conception and design. YA, YK, NM and KO were involved in acquisition of data. YA, YT, YK, NM, KO, and RMH analyzed and interpreted data. YA and RMH wrote the manuscript. All Authors reviewed and approved the manuscript.
Conflicts of Interest
The Authors declare that there are no conflicts of interest to declare in relation to this study.
Funding
This study was funded, in part, by the Robert M. Hoffman Foundation for Cancer Research and by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Early-Career Scientists (24K19627).
- Received August 7, 2024.
- Revision received September 3, 2024.
- Accepted September 4, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
