Open Access

Synergy of Rapamycin and Methioninase on Colorectal Cancer Cells Requires Simultaneous and Not Sequential Administration: Implications for mTOR Inhibition

ARDJMAND DANIEL 1
SATO MOTOKAZU 1 2
HAN QINGHONG 1
KUBOTA YUTARO 1 2
MIZUTA KOHEI 1 2
MORINAGA SEI 1 2
  &  
HOFFMAN ROBERT M. 1 2

1AntiCancer Inc., San Diego, CA, U.S.A.

2Department of Surgery, University of California San Diego, San Diego, CA, U.S.A.

Cancer Diagnosis & Prognosis Jul-Aug; 4(4): 396-401 DOI: 10.21873/cdp.10338
Received 26 February 2024 | Revised 13 July 2024 | Accepted 05 April 2024
Corresponding author
Robert M. Hoffman, Ph.D., AntiCancer Inc, 7917 Ostrow St, San Diego, CA, 92111, U.S.A. Tel: +1 6198852284, email: all@anticancer.com

Abstract

Background/Aim: Rapamycin inhibits the mTOR protein kinase. Methioninase (rMETase), by degrading methionine, targets the methionine addiction of cancer cells and has been shown to improve the efficacy of chemotherapy drugs, reducing their effective doses. Our previous study demonstrated that rapamycin and rMETase work synergistically against colorectal-cancer cells, but not on normal cells, when administered simultaneously in vitro. In the present study, we aimed to further our previous findings by exploring whether  synergy exists between rapamycin and rMETase when used sequentially against HCT-116 colorectal-carcinoma cells, compared to simultaneous administration, in vitro. Materials and Methods: The half-maximal inhibitory concentrations (IC50) of rapamycin alone and rMETase alone against the HCT-116 human colorectal-cancer cell line were previously determined using the CCK-8 cell viability assay (11). We then examined the efficacy of rapamycin and rMETase, both at their IC50, administered simultaneously or sequentially on the HCT-116 cell line, with rapamycin administered before rMETase and vice versa. Results: The IC50 for rapamycin and rMETase, determined from previous experiments (11), was 1.38 nM and 0.39 U/ml, respectively, of HCT-116 cells. When rMETase was administered four days before rapamycin, both at the IC50, there was a 30.46% inhibition of HCT-116 cells. When rapamycin was administered four days before rMETase, both at the IC50, there was an inhibition of 41.13%. When both rapamycin and rMETase were simultaneously administered, both at the IC50, there was a 71.03% inhibition. Conclusion: Rapamycin and rMETase have synergistic efficacy against colorectal-cancer cells in vitro when administered simultaneously, but not sequentially.
Keywords: methioninase, rMETase, rapamycin, mTOR, SAMTOR, SAM, combination, simultaneous, sequential, synergy, cancer cells, IC50, HCT-116, Methionine addiction, Hoffman effect

Mammalian target of rapamycin (mTOR) is a serine-threonine protein kinase that regulates critical aspects of cellular metabolism. Rapamycin (sirolimus) and its analogs, such as temsirolimus and everolimus, are inhibitors of mTOR and have shown modest efficacy against various cancers (1).

mTOR (mTORC1) has been shown to regulate glycolysis, glutamine metabolism, autophagy, and its activity partially depends on sensing methionine. The methionine metabolite S-adenosylmethionine (SAM) activates mTOR by binding SAMTOR (2). The cellular concentration of SAM is rapidly reduced by methionine restriction of cancer cells (3), which are methionine addicted, due to overuse of methionine and SAM for abnormally-elevated transmethylation reactions (4-8).

To target the methionine addiction of cancer cells, recombinant methioninase (rMETase), cloned from Pseudomonas putida into E. Coli, is used to degrade methionine (9). We have previously shown that rMETase combined with rapamycin synergistically eradicated an osteosarcoma of the breast, in a patient-derived orthotopic xenograft (PDOX) mouse model without toxicity (10). The combination of rMETase and rapamycin administered simultaneously also demonstrated great synergy on HCT-116 colorectal-carcinoma cells, but not normal fibroblasts, in vitro (11). These results suggest that an acute deficiency of SAM, effected by methionine restriction, in cancer cells, in combination with rapamycin, greatly inhibits mTOR’s protein kinase activity, preventing cancer-cell proliferation (3,12).

In the present study, we administered rapamycin and rMETase both simultaneously and sequentially to human colorectal carcinoma cells (HCT-116) in vitro to determine whether the synergy of these two agents depends on the timing of their administration. The results suggest a possible novel mechanism of mTOR inhibition.

Materials and Methods

Cell culture. The HCT-116 human colon cancer cell line (American Type Culture Collection Manassas, VA, USA) was grown in Dulbecco’s modified Eagles’ medium (DMEM) with 10% fetal bovine serum and 100 IU/ml of penicillin/streptomycin.

rMETase production and formulation. rMETase was produced at AntiCancer Inc. (San Diego, CA, USA). Escherichia coli was previously transformed with the methioninase gene from Pseudomonas putida and fermented (9). rMETase was purified from recombinant E. Coli with a 60˚C heat step, precipitation with polyethylene-glycol, and final purification with diethylaminoethyl-sepharose fast-flow ion-exchange column chromatography (9).

Cell viability testing. HCT-116 cells were cultured at subconfluence overnight in DMEM in 96-well plates (1.0×103 cells per well). The following day, HCT-116 cells were treated with IC50 concentrations of rapamycin (IC50=1.38 nM [11]) or rMETase (IC50=0.39 U/ml [11]), either simultaneously or sequentially. HCT-116 cells were treated for eight days with rMETase or rapamycin alone. The HCT-116 cells were treated for 8 days with the simultaneous combination of rMETase and rapamycin. For sequential treatment, HCT-116 cells were treated for four days with rMETase first, followed by a wash with phosphate-buffered saline (PBS), and then treated with rapamycin for another four days, or vice versa. Cell viability was determined with the Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan) using the WST-8 reagent.

ImageJ version 1.53 (National Institutes of Health, Bethesda, MD, USA) was applied to produce IC50 and sensitivity curves. IC50 values were calculated from the raw data. Each experiment was carried out in triplicate.

Statistics. GraphPad Prism 9.4.0 (GraphPad Software, Inc., San Diego, CA, USA) was used to conduct all statistical analyses. Tukey’s multiple comparison test was performed for the parametric test of comparison between groups. All data are presented as the mean and standard deviation. The significance level was set at p≤0.05.

Results

The IC50 of HCT-116 cells for rapamycin alone and rMETase alone was 1.38 nM (11) and 0.39 U/ml (11), respectively (Figure 1). The IC50 of rMETase alone significantly inhibited the HCT-116 cells (p=0.0048) but the IC50 of rapamycin alone did not significantly inhibit the HCT-116 cells (p=0.4032) (Figure 2). Figures 1 and 2 are from independent experiments. When both rapamycin and rMETase were simultaneously administered, both at the IC50, there was a 71.03% inhibition (Table I, Table II, Figure 3, Figure 4). When rMETase was administered four days before rapamycin, both at the IC50, there was a 30.5% inhibition (Table I, Table II, Figure 3, Figure 4). When rapamycin was administered for four days before rMETase, both at the IC50, there was an inhibition of 41.1% (Table I, Table II, Figure 3, Figure 4).

Discussion

Methionine addiction is termed the Hoffman Effect and is a fundamental hallmark of cancer (4,12-19). Due to methionine addiction, cancer cells are inhibited by methionine restriction, which severely depletes methionine and SAM in the cancer cells (3,4,12). rMETase indirectly inhibits mTOR activity by acute depletion of methionine (MET) which depletes SAM in cancer cells resulting in SAMTOR binding to GATOR instead of SAM, thereby inhibiting mTOR (3) (Figure 4B, Figure 4C). Rapamycin forms an inhibitory complex with FKBP12 to block the kinase activity of mTOR (Figure 4A, Figure 4C). Rapamycin and rMETase when used simultaneously have synergistic efficacy against HCT-116 human colorectal-cancer cells in vitro (11) as well as against osteosarcoma of the breast in vivo (10). As shown in the present study, synergy was not observed when rapamycin and methioninase were administered sequentially (Table I, Table II, Figure 3, Figure 4). These results suggest that the synergistic inhibition of mTOR requires rapamycin and rMETase to be simultaneously present (Table I, Table II, Figure 3, Figure 4). Further studies are needed to describe the mechanism in detail. The present in vitro result and our previous in vitro (11) and in vivo results (10) showing synergy of rapamycin and methioninase against cancer cells and not normal cells, suggest that this combination has the potential for future clinical use when administered simultaneously as it targets a fundamental hallmark of cancer (1-6,11-19,20-31), methionine addiction, known as the Hoffman effect (16,18,19).

Conflicts of Interest

The Authors have no conflicts of interest to declare in relation to this study.

Authors’ Contributions

DA performed experiments. QH supplied methioninase. DA and RMH contributed the concept of the study and wrote the manuscript. DA and RMH revised the manuscript. DA, YK, MS, QH, KM, SM, and RMH critically read the manuscript.

Acknowledgements

This paper is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Professor Gordon H. Sato, Professor Li Jiaxi, Masaki Kitajima, MD, Shigeo Yagi, PhD, Jack Geller, MD, Joseph R. Bertino, MD, J.A.R. Mead, PhD. Professor Sheldon Penman and Professor John R. Raper. The Robert M. Hoffman Foundation for Cancer Research provided funds for this study.

References

1 Li J Kim SG & Blenis J Rapamycin: one drug, many effects. Cell Metab. 19(3) 373 - 379 2014. DOI: 10.1016/j.cmet.2014.01.001
2 Gu X Orozco JM Saxton RA Condon KJ Liu GY Krawczyk PA Scaria SM Harper JW Gygi SP & Sabatini DM SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway. Science. 358(6364) 813 - 818 2017. DOI: 10.1126/science.aao3265
3 Coalson DW Mecham JO Stern PH & Hoffman RM Reduced availability of endogenously synthesized methionine for S-adenosylmethionine formation in methionine-dependent cancer cells. Proc Natl Acad Sci USA. 79(14) 4248 - 4251 1982. DOI: 10.1073/pnas.79.14.4248
4 Wang Z Yip LY Lee JHJ Wu Z Chew HY Chong PKW Teo CC Ang HY Peh KLE Yuan J Ma S Choo LSK Basri N Jiang X Yu Q Hillmer AM Lim WT Lim TKH Takano A Tan EH Tan DSW Ho YS Lim B & Tam WL Methionine is a metabolic dependency of tumor-initiating cells. Nat Med. 25(5) 825 - 837 2019. DOI: 10.1038/s41591-019-0423-5
5 Stern PH & Hoffman RM Elevated overall rates of transmethylation in cell lines from diverse human tumors. In Vitro. 20(8) 663 - 670 1984. DOI: 10.1007/BF02619617
6 Yamamoto J Aoki Y Han Q Sugisawa N Sun YU Hamada K Nishino H Inubushi S Miyake K Matsuyama R Bouvet M Endo I & Hoffman RM Reversion from methionine addiction to methionine independence results in loss of tumorigenic potential of highly-malignant lung-cancer cells. Anticancer Res. 41(2) 641 - 643 2021. DOI: 10.21873/anticanres.14815
7 Ghergurovich JM Xu X Wang JZ Yang L Ryseck RP Wang L & Rabinowitz JD Methionine synthase supports tumour tetrahydrofolate pools. Nat Metab. 3(11) 1512 - 1520 2021. DOI: 10.1038/s42255-021-00465-w
8 Sullivan MR Darnell AM Reilly MF Kunchok T Joesch-Cohen L Rosenberg D Ali A Rees MG Roth JA Lewis CA & Vander Heiden MG Methionine synthase is essential for cancer cell proliferation in physiological folate environments. Nat Metab. 3(11) 1500 - 1511 2021. DOI: 10.1038/s42255-021-00486-5
9 Tan Y Xu M Tan X Tan X Wang X Saikawa Y Nagahama T Sun X Lenz M & Hoffman RM Overexpression and large-scale production of recombinantl-methionine-α-deamino-γ-mercapto methane-lyase for novel anticancer therapy. Protein Expr Purif. 9(2) 233 - 245 1997. DOI: 10.1006/prep.1996.0700
10 Masaki N Han Q Samonte C Wu NF Hozumi C Wu J Obara K Kubota Y Aoki Y Bouvet M & Hoffman RM Oral-recombinant methioninase in combination with rapamycin eradicates osteosarcoma of the breast in a patient-derived orthotopic xenograft mouse model. Anticancer Res. 42(11) 5217 - 5222 2022. DOI: 10.21873/anticanres.16028
11 Ardjmand D Kubota Y Sato M Han Q Mizuta K Morinaga S & Hoffman RM Selective synergy of rapamycin combined with methioninase on cancer cells compared to normal cells. Anticancer Res. 44(3) 929 - 933 2024. DOI: 10.21873/anticanres.16887
12 Stern PH Mecham JO Wallace CD & Hoffman RM Reduced free-methionine in methionine-dependent SV40-transformed human fibroblasts synthesizing apparently normal amounts of methionine. J Cell Physiol. 117(1) 9 - 14 1983. DOI: 10.1002/jcp.1041170103
13 Hoffman RM & Erbe RW High in vivo rates of methionine biosynthesis in transformed human and malignant rat cells auxotrophic for methionine. Proc Natl Acad Sci USA. 73(5) 1523 - 1527 1976. DOI: 10.1073/pnas.73.5.1523
14 Sugimura T Birnbaum SM Winitz M & Greenstein JP Quantitative nutritional studies with water-soluble, chemically defined diets. VIII. The forced feeding of diets each lacking in one essential amino acid. Arch Biochem Biophys. 81(2) 448 - 455 1959. DOI: 10.1016/0003-9861(59)90225-5
15 Yamamoto J Han Q Inubushi S Sugisawa N Hamada K Nishino H Miyake K Kumamoto T Matsuyama R Bouvet M Endo I & Hoffman RM Histone methylation status of H3K4me3 and H3K9me3 under methionine restriction is unstable in methionine-addicted cancer cells, but stable in normal cells. Biochem Biophys Res Commun. 533(4) 1034 - 1038 2020. DOI: 10.1016/j.bbrc.2020.09.108
16 Kaiser P Methionine Dependence of Cancer. Biomolecules. 10(4) 568 2020. DOI: 10.3390/biom10040568
17 Mecham JO Rowitch D Wallace CD Stern PH & Hoffman RM The metabolic defect of methionine dependence occurs frequently in human tumor cell lines. Biochem Biophys Res Commun. 117(2) 429 - 434 1983. DOI: 10.1016/0006-291x(83)91218-4
18 Guo R Liang JH Zhang Y Lutchenkov M Li Z Wang Y Trujillo-Alonso V Puri R Giulino-Roth L & Gewurz BE Methionine metabolism controls the B cell EBV epigenome and viral latency. Cell Metab. 34(9) 1280 - 1297.e9 2022.
19 Bin P Wang C Zhang H Yan Y & Ren W Targeting methionine metabolism in cancer: opportunities and challenges. Trends Pharmacol Sci. 5 S0165 - 6147(24)00050-6 2024. DOI: 10.1016/j.tips.2024.03.002
20 Hoffman RM Jacobsen SJ & Erbe RW Reversion to methionine independence in simian virus 40-transformed human and malignant rat fibroblasts is associated with altered ploidy and altered properties of transformation. Proc Natl Acad Sci USA. 76(3) 1313 - 1317 1979. DOI: 10.1073/pnas.76.3.1313
21 Hoffman RM Jacobsen SJ & Erbe RW Reversion to methionine independence by malignant rat and SV40-transformed human fibroblasts. Biochem Biophys Res Commun. 82(1) 228 - 234 1978. DOI: 10.1016/0006-291x(78)90600-9
22 Kubota Y Sato T Hozumi C Han Q Aoki Y Masaki N Obara K Tsunoda T & Hoffman RM Superiority of [(11)C]methionine over [(18)F]deoxyglucose for PET imaging of multiple cancer types due to the methionine addiction of cancer. Int J Mol Sci. 24(3) 1935 2023. DOI: 10.3390/ijms24031935
23 Stern PH & Hoffman RM Enhanced in vitro selective toxicity of chemotherapeutic agents for human cancer cells based on a metabolic defect. J Natl Cancer Inst. 76(4) 629 - 639 1986. DOI: 10.1093/jnci/76.4.629
24 Hoffman RM Coalson DW Jacobsen SJ & Erbe RW Folate polyglutamate and monoglutamate accumulation in normal and SV40-transformed human fibroblasts. J Cell Physiol. 109(3) 497 - 505 1981. DOI: 10.1002/jcp.1041090316
25 Aoki Y Han Q Tome Y Yamamoto J Kubota Y Masaki N Obara K Hamada K Wang JD Inubushi S Bouvet M Clarke SG Nishida K & Hoffman RM Reversion of methionine addiction of osteosarcoma cells to methionine independence results in loss of malignancy, modulation of the epithelial-mesenchymal phenotype and alteration of histone-H3 lysine-methylation. Front Oncol. 12 1009548 2022. DOI: 10.3389/fonc.2022.1009548
26 Yamamoto J Inubushi S Han Q Tashiro Y Sugisawa N Hamada K Aoki Y Miyake K Matsuyama R Bouvet M Clarke SG Endo I & Hoffman RM Linkage of methionine addiction, histone lysine hypermethylation, and malignancy. iScience. 25(4) 104162 2022. DOI: 10.1016/j.isci.2022.104162
27 Tan Y Xu M & Hoffman RM 27Tan Y, Xu M, Hoffman RM: Broad selective efficacy of recombinant methioninase and polyethylene glycol-modified recombinant methioninase on cancer cells In Vitro. Anticancer Res. 30(4) 1041 - 6 2010.
28 Yamamoto J Aoki Y Inubushi S Han Q Hamada K Tashiro Y Miyake K Matsuyama R Bouvet M Clarke SG Endo I & Hoffman RM Extent and instability of trimethylation of histone H3 lysine increases with degree of malignancy and methionine addiction. Cancer Genomics Proteomics. 19(1) 12 - 18 2022. DOI: 10.21873/cgp.20299
29 Aoki Y Han Q Kubota Y Masaki N Obara K Tome Y Bouvet M Nishida K & Hoffman RM Oncogenes and methionine addiction of cancer: Role of c-MYC. Cancer Genomics Proteomics. 20(2) 165 - 170 2023. DOI: 10.21873/cgp.20371
30 Stern PH Mecham JO Wallace CD & Hoffman RM Reduced free-methionine in methionine-dependent SV40-transformed human fibroblasts synthesizing apparently normal amounts of methionine. J Cell Physiol. 117(1) 9 - 14 1983. DOI: 10.1002/jcp.1041170103
31 Jacobsen SJ Hoffman RM & Erbe RW Regulation of methionine adenosyltransferase in normal diploid and simian virus 40-transformed human fibroblasts. J Natl Cancer Inst. 65(6) 1237 - 1244 1980.