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.

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.

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).

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).

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

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.

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.