Abstract
Background: Methionine dependence may be the only known general metabolic defect in cancer. In order to exploit methionine dependence for therapy, our laboratory previously cloned L-methionine α-deamino-γ-mercaptomethane lyase [EC 4.4.1.11]) (recombinant methioninase [rMETase]), which was subsequently tested in mouse models of various types of human tumors. The present study aimed to investigate the efficacy of rMETase on human osteosarcoma cells in vitro and in vivo. Materials and Methods: Human osteosarcoma cell lines 143B, HOS and SOSN2 were tested in vitro for survival during a 72-h exposure to rMETase using the WST-8 assay. Half-maximal inhibitory concentrations were calculated for in vitro efficacy experiments. 143B cells were orthotopically transplanted into the tibia of nude mice. Mouse models were randomized into the following groups 1 week after transplantation: Group 1, untreated control; Group 2, cisplatinum (CDDP) [intraperitoneal (i.p.) injection at 6 mg/kg weekly, for 3 weeks], positive control; Group 3, rMETase, 100 units/mouse i.p. daily, for 21 days. Tumor sizes and body weight were measured with calipers and a digital balance once per week, respectively. Results: rMETase significantly inhibited osteosarcoma cell growth, in a dose-dependent manner, in vitro. Both CDDP and rMETase treatment significantly inhibited tumor volume compared to untreated control mice at 5 weeks after initiation. Tumor volumes were as follows: Group 1, untreated, control: 1808.2 ± 344 mm3; Group 2, CDDP: 1102.2 ± 316 mm3, p=0.0008 compared to untreated control; Group 3, rMETase: 884.8 ± 361 mm3, p=0.0001 compared to untreated control. There were no animal deaths in any group. The body weight of mice was not significantly different between any group. Conclusion: rMETase showed promising efficacy against osteosarcoma, a recalcitrant tumor type. Future studies will investigate the efficacy of rMETase on patient-derived orthotopic xenograft (PDOX) models of osteosarcoma as a bridge to testing rMETase in the clinic.
- Metabolic targeting
- osteosarcoma
- recombinant methioninase
- rMETase
- treatment
- in vitro
- orthotopic
- nude mice
- efficacy
Methionine (MET) dependence may be the only known general and very widespread metabolic defect in cancer (1). Numerous cell types have been shown to arrest their growth when deprived of MET (2-6). Malignant human cell lines were able to synthesize MET from homocysteine at rates that were at least as high as normal human skin fibroblast strains (7). These data suggest that MET dependence may be caused by an altered and enhanced utilization of MET as opposed to an inability to synthesize MET from homocysteine (7). Methionine deprivation causes cancer cells to arrest predominantly in the S/G2 phases of the cell cycle and to eventually undergo apoptosis (6-11).
We previously observed that cancer cells have enhanced overall rates of transmethylation compared to normal human fibroblasts (12). The overuse of methionine for enhanced and unbalanced transmethylation may be the basis of the methionine dependence of cancer cells. The overuse of methionine by cancer cells is termed the ‘Hoffman effect’ (13-15). The alteration of such a fundamental process as transmethylation in cancer may be indicative of its importance in the oncogenic process (12-14).
Methionine depletion can be achieved using recombinant L-methioninedeamino-γ-mercaptomethane-lyase [EC 4.4.1.11] (recombinant methioninase [rMETase], a methionine-cleaving enzyme derived from Pseudomonas putida) (16). Our laboratory previously cloned and expressed rMETase in Escherichia coli (17).
Previously, 21 different human cancer cell lines (four each of lung, colon, kidney, and melanoma, three from CNS, and two from prostate) and normal cell strains were treated with rMETase in vitro. rMETase had a mean half-maximal inhibitory concentration (IC50) for cancer cells which was one order of magnitude lower than that for normal cell strains (4).
rMETase arrested growth of HCT 15 and HT29 colon cancer in nude mice for 1 week after treatment termination. Growth of Colo 205 and SW 620 colon cancers were partially arrested by rMETase (18). Methionine dependence was also found to occur in fresh patient tumors histocultured on Gelfoam® (5, 6). rMETase also arrested growth of a Ewing's sarcoma patient-derived orthotopic xenograft (PDOX) mouse model (15).
Osteosarcoma is a common malignant primary bone tumor occurring in patients aged 10-25 years. Osteosarcoma has a 5-year survival rate of approximately 70% in patients treated with chemotherapy and surgery. However, the survival rate is much lower after metastasis has occurred (19-21). The most effective chemotherapeutic drugs against osteosarcoma are high-dose methotrexate, cisplatinum, doxorubicin, and ifosfamide. However, dose escalation of these drugs did not improve the outcome of osteosarcoma patients (22). Since the survival of patients with osteosarcoma has plateaued, transformational new treatment approaches are needed.
In the present study, we evaluated the efficacy of rMETase on human osteosarcoma cell lines in vitro and in vivo in an orthotopic mouse model as a potential treatment for clinical osteosarcoma in the future.
Materials and Methods
Cell lines and growth conditions. The following human osteosarcoma cell lines were used: 143B, HOS and SOSN2 (23, 24). Cells were kept in log phase by supplementation with fresh medium 2-3 times/week. All cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.
Growth-inhibition assay. Cell viability was assessed using the WST-8 dye reduction assay. Briefly, cells were seeded in 96-well flat-bottomed microplates (100 μlmedium/well) at a density of 5×104 cells/ml, incubated at 37°C for 24 h, and exposed to different concentrations of tested compounds for 72 h. For each drug concentration, at least 8 wells were used. Before the WST-8 assay was performed, cells were observed with the Power IX71 microscope (Olympus Corp., Tokyo, Japan) at ×200 magnification. After incubation with rMETase, 10 μl WST-8 solution was added to each well. The microplates were further incubated for 3 h at 37°C, and absorption was measured using a microprocessor-controlled microplate reader (iMark™; Bio-Rad Laboratories, Hercules, CA, USA) at 450 nm. Cell-survival fractions were calculated as a percentage of untreated-control cells. Half-maximal inhibitory concentration (IC50) values were derived from concentration–response curves.
Mice. Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA, USA), 4-6 weeks old, were used in this study. Animals were housed in a barrier facility on a high-efficiency particulate arrestance-filtered rack under standard conditions of 12-h light/dark cycles. The animals were fed an autoclaved laboratory rodent diet. All animal studies were conducted with an AntiCancer Institutional Animal Care and Use Committee protocol specifically approved for this study and in accordance with the principles and procedures outlined in the National Institute 5 of Health Guide for the Care and Use of Animals under Assurance Number A3873-1. In order to minimize any suffering of the animals, anesthesia and analgesics were used for all surgical experiments. The response of animals during surgery was monitored to ensure adequate depth of anesthesia. The animals were observed on a daily basis and humanely sacrificed by CO2 inhalation when they met the following humane endpoint criteria: severe tumor burden (more than 20 mm in diameter), prostration, significant-body weight loss, difficulty inbreathing, rotational motion and drop in body temperature (1).
Intra-tibial 143B transplantation. The mice were anesthetized by subcutaneous injection of a ketamine mixture (0.02 ml solution of 20 mg/kg ketamine, 15.2 mg/kg xylazine and 0.48 mg/kg acepromazine maleate). A skin incision was made for tibial exposure. A pin hole was made in the proximal tibia. Then, a suspension of 143B cells (2×105) in 10 μl phosphate-buffered saline containing 5 μg Matrigel™ (Becton Dickinson, Bedford, MA, USA) to prevent the suspension from leaking out, was transplanted through the pinhole using a 23-gauge needle. Tumors were measured once weekly using digital calipers and weighed (24-27).
One week after transplantation, mouse models were randomized into the following groups (n=8): Group 1, control without treatment; Group 2, cisplatinum (CDDP): 6 mg/kg, intraperitoneal (i.p.), weekly, for 3 weeks; Group 3, rMETase, 100 units/mouse i.p. daily, for 21 days (18). Tumor sizes and body weight were measured with calipers and digital balance, respectively, once a week (Figure 1).
Primary tumor growth measurement. The tumors were allowed to form and tumor dimensions were measured weekly. Tumor volumes were calculated using the following equation: volume=4π (A/2)(B/2)(C/2)/3, where A is the width (average distance in the medial–lateral plane), B is the length (average distance in the proximal–distal plane), and C is the width (average distance in the anterior–posterior plane).
Results and Discussion
Cytotoxicity. The cytotoxic activity of rMETase was determined against 143B, HOS, and SOSN2 osteosarcoma cell lines, which were incubated for 72 h with rMETase or CDDP. Cell survival was evaluated as described in the Materials and Methods. rMETase significantly inhibited osteosarcoma cell growth in a dose-dependent manner (Figures 2 and 3, Table I). There was approximately a 7-fold range in the sensitivity of osteosarcoma cells, with 143B being the most sensitive and SOSN2 being the least sensitive.
Orthotopic mouse model. 143B cells were transplanted intratibially in nude mice. One week after transplantation, mice were treated with either rMETase or CDDP In both treatment groups, osteosarcoma growth was significantly inhibited compared to untreated controls. At 5 weeks after initiation of treatment, tumor volumes were as follows: control: 1808.2 ± 344 mm3; CDDP: 1102.2 ± 316 mm3, p=0.0008; rMETase: 884.8 ± 361 mm3, p=0.0001 (Figure 4).
There were no animal deaths in any group. The body weight of mice was not significantly different in any group (Figure 5).
The first indication that methionine metabolism is perturbed in cancer was presented by Sugimura et al., almost 60 years ago, who observed that rat tumor growth was slowed by a diet depleted of methionine (28). Subsequently, L5178Y mouse leukemia cells in culture were observed to require very high levels of methionine in order to proliferate (29). Many cancer cell lines were then found to be methionine dependent, requiring high amounts of methionine in order to proliferate (3, 4). Tumors from human patients, including tumors of the colon, breast, ovary, prostate, and a melanoma, were also found to be methionine-dependent in Gelfoam® histoculture (5). The occurrence of methionine dependence among diverse cancer types indicates methionine dependence may be a general phenomena in cancer. In contrast, normal unestablished cell strains characterized grew well in methionine-depleted medium (3).
Methionine restriction selectively arrests cancer cells in the S/G2 phases of the cell cycle (6, 8, 10, 11), which can be exploited for effective chemotherapy (9-11).
A recent article appeared in 2017 with the title “The new anticancer era: Tumor metabolism targeting” (30). However, this “new anticancer era” started in 1959 with the observation of Sugimura et al. that depriving cancer of methionine arrested tumor growth (28). The current results and previous reports demonstrate that targeting methionine metabolism has great potential for cancer therapy. Our current and previous (15) findings suggest an important future clinical target for rMETase in osteosarcoma.
Footnotes
Dedication
This article is dedicated to the memory of A. R. Moossa, M.D., and Sun Lee, M.D.
- Received June 9, 2017.
- Revision received June 29, 2017.
- Accepted July 3, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved