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Primary osteosarcoma of the breast is a very rare malignancy that shares histological features with osteosarcoma. It is also highly sensitive to methionine restriction due to methionine addiction. We previously established a patient-derived orthotopic xenograft (PDOX) nude-mouse model derived from tumor tissue of a patient with primary mammary osteosarcoma. In the present study, we investigated the efficacy of oral-recombinant methioninase (o-rMETase), combined with rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on a mammary osteosarcoma PDOX nude-mouse model.The PDOX mouse model was established by surgically transplanting a specimen of primary osteosarcoma of the breast into the mammary gland of nude mice. Mice implanted with tumors were randomly divided into four groups: Control group, N=5; rapamycin-treated group, N=5; o-rMETase-treated group, N=5; and a group treated with the combination of o-rMETase and rapamycin, N=5. Mice were treated for 2 weeks after transplantation, and tumor volume was measured during the treatment period.Treatment with the combination of rapamycin and o-rMETase eradicated the osteosarcoma of the breast compared to the untreated control (p=0.000008). o-rMETase alone did not significantly inhibit tumor growth, and rapamycin alone only partially inhibited the tumor (p=0.78 and p=0.018, respectively) compared to the untreated control. There was not a significant difference in mouse weight between the groups.The combination of rapamycin and o-rMETase was highly effective against primary osteosarcoma of the breast in a PDOX model, suggesting a future clinical strategy for this rare cancer type that currently has no first-line treatment.

Rectal cancer is a recalcitrant disease with limited treatment options. Pre-clinical studies have shown the efficacy of methionine restriction with a low-methionine diet and oral recombinant methioninase (o-rMETase) for colorectal cancer. There are also clinical studies on methionine restriction with o-rMETase for other recalcitrant cancer types. The goal of the present study was to determine the efficacy of a low-methionine diet and o-rMETase on a rectal cancer patient.A 55-year-old man diagnosed with recurrent locally-advanced rectal-cancer was treated with o-rMETase and a low-methionine diet, during which time, he did not receive standard chemotherapy. Disease stability was monitored by carcinoembryonic antigen (CEA) levels, sigmoidoscopy, and computed tomography (CT).The patient was diagnosed with stage II rectal cancer (adenocarcinoma) in 2018. After neoadjuvant chemoradiotherapy, the patient received total mesorectal excision (TME) in 2018. Local recurrence was found by sigmoidoscopy one year later. The patient was given chemotherapy, the recurrent lesion shrunk, and was then removed endoscopically in December 2019, with positive margins. The tumor did not become apparent for about a year after that. An endoscopic examination performed in December 2020, revealed a local recurrence. Since that time, the patient had an elevated CEA. The patient went on o-rMETase and a low-methionine diet from January 2021. Since then, the patient's CEA level has remained stable for the next year and a half. He received sigmoidoscopy and CT regularly, and the tumor size has not changed.This patient's clinical course indicates that o-rMETase and a low-methionine diet may be effective for rectal cancer, for long-term disease stabilization. Further case studies and clinical trials are needed to determine the generality of the present result.

All cancer cell types are methionine-addicted, which is termed the Hoffman effect. Cancer cells, unlike normal cells, cannot survive without large amount of methionine. In general, when methionine is depleted, both normal cells and cancer cells synthesize methionine from homocysteine, but cancer cells consume large amounts of methionine and they cannot survive without exogenous methionine. For this reason, methionine restriction has been shown to be effective against many cancers in vitro and in vivo. Methionine restriction arrests cancer cells in the S/G2-phase of the cell cycle. Cytotoxic agents that act in the S/G2-phase are highly effective when used in combination with methionine restriction due to the cancer cells being trapped in S/G2-phase, unlike normal cells which arrest in G1/G0-phase. Combining methionine restriction and chemotherapeutic drugs for cancer treatment is termed the Hoffman protocol. The efficacy of many cytotoxic agents and molecular-targeted drugs in combination with methionine restriction has been demonstrated. The most effective method of methionine restriction is the administration of recombinant methioninase (rMETase), which degrades methionine. The efficacy of rMETase has been reported in mice and human patients by oral administration. The present review describes studies on anticancer drugs that showed synergistic efficacy in combination with methionine restriction, including rMETase administration. It is proposed that the next disruptive generation of cancer chemotherapy should employ current therapy in combination with methionine restriction for all cancer types.

Background/Aim: Methionine addiction is a general and fundamental hallmark of cancer cells, termed the Hoffman effect. Previously Vanhamme and Szpirer showed that methionine addiction could be induced by transfection of the activated HRAS1 gene to a normal cell line. In the present study, we investigated the role of the c-MYC oncogene in methionine addiction of cancer, by comparison of c-Myc expression and malignancy of methionine-addicted osteosarcoma cells and rare methionine-independent revertants, derived from the methionine-addicted cells. Materials and Methods: Methionine-independent revertant 143B osteosarcoma cells (143B-R) were derived from methionine-addicted parental 143B osteosarcoma cells (143B-P), by continuous culture in medium depleted of methionine by recombinant methioninase. To compare in vitro malignancy of methionine-addicted parental cells and methionine-independent revertant cells, the following experiments were performed: for 143B-P and 143B-R cells, cell proliferation capacity was measured with a cell-counting assay, and colony-formation capacity was determined on plastic and in soft agar, all in methionine-containing Dulbecco’s Modified Eagle’s Medium (DMEM). Tumor growth was measured in orthotopic xenograft nude-mouse models, to compare in vivo malignancy of 143B-P and 143B-R cells. c-MYC expression was examined with western immunoblotting and compared in 143B-P and 143B-R cells. Results: 143B-R cells had reduced cell proliferation capacity, compared to 143B-P cells, in methionine-containing medium (p=0.003). 143B-R cells had reduced colony formation capacity on plastic (p=0.003) and in soft agar, compared to 143B-P cells in methionine-containing medium. 143B-R cells had reduced tumor growth in orthotopic xenograft nude-mouse models, compared to 143B-P cells, (p=0.002). These results demonstrate that 143B-R methionine-independent revertant cells lost malignancy. Expression of c-MYC was reduced in 143B-R methionine-independent revertant osteosarcoma cells, compared to 143B-P cells, (p=0.0007). Conclusion: The present study demonstrated that c-MYC expression is linked to malignancy and methionine addiction of cancer cells. The present study on c-MYC, and the previous study on HRAS1, suggest that oncogenes may play a role in methionine addiction, which is a hallmark of all cancers, as well as in malignancy.

Osteosarcoma is the most common bone sarcoma. Although surgery and chemotherapy are initially effective, the 5-year survival is approximately 60% to 80%, and has not improved over three decades. We have previously shown that methionine restriction (MR) induced by oral recombinant methioninase (o-rMETase), is effective against osteosarcoma in patient-derived orthotopic xenograft (PDOX) nude-mouse models. In the present report, the efficacy of the combination of oral o-rMETase and methotrexate (MTX) was examined in an osteosarcoma PDOX mouse model.An osteosarcoma-PDOX model was previously established by implanting tumor fragments into the proximal tibia of nude mice. The osteosarcoma PDOX models were randomized into four groups: control; o-rMETase alone; MTX alone; combination of o-rMETase and MTX. The mice were sacrificed after 4 weeks of treatment.The combination of o-rMETase and MTX showed significantly higher efficacy compared to the control group (p=0.04). The combination also showed significantly higher efficacy compared to MTX alone (p=0.04). No significant efficacy of o-rMETase alone or MTX alone compared to control was shown (p=0.21, 1.00, respectively). Only the combination of o-rMETase and MTX reduced the cancer-cell density in the osteosarcoma tumor.rMETase converted an osteosarcoma PDOX from MTX-resistant to MTX-sensitive and thereby shows future clinical potential.

Attempts to selectively starve cancers in the clinic have been made at least since the time of Warburg beginning 100 years ago. Calorie-restriction or low-carbohydrate diets have had limited success with cancer patients. Methionine restriction is another strategy to selectively starve cancer cells, since cancers are addicted to methionine, unlike normal cells. Methionine addiction of cancer is termed the Hoffman effect. Numerous preclinical studies over the past half century have shown methionine restriction to be highly effective against all major cancer types and synergistic with chemotherapy. Low-methionine medical diets can be effective in lowering methionine and have shown some clinical promise, but they are not palatable and thereby not sustainable. However, selectively choosing among plant-based foods allows a variety of low-methionine diets that are sustainable. Our laboratory has developed a methioninase that can be administered orally as a supplement and has resulted in anecdotal positive results in patients with advanced cancer, including hormone-independent prostate cancer, and other recalcitrant cancers. The question is whether methionine restriction through a low-methionine diet, or even greater methionine restriction with methioninase in combination with a low-methionine diet, is ready for prime time in the clinic, especially in combination with other synergistic therapy. The question will hopefully be answered in the near future, especially for advanced cancer patients who have failed all standard therapy.

Gastric cancer is highly malignant and recalcitrant to first line chemotherapies that include 5-fluorouracil (5-FU). Cancer cells are addicted to methionine for their proliferation and survival. Methionine addiction of cancer is known as the Hoffman effect. Methionine restriction with recombinant methioninase (rMETase) has been shown to selectively starve cancer cells and has shown synergy with cytotoxic chemotherapy including 5-FU. The present study aimed to investigate the efficacy of rMETase alone and the combination with 5-FU on poorly differentiated human gastric cancer cell lines (MKN45, NUGC3, and NUGC4) in vitro and vivo. rMETase suppressed the tumor growth of 3 kinds of poorly differentiated gastric cancer cells in vitro. The fluorescence ubiquitination-based cell cycle indicator (FUCCI) demonstrated cancer cells treated with rMETase were selectively trapped in the S/G

Background/Aim: Primary osteosarcoma of the mammary gland is a very rare disease, accounting for under 1% of all mammary gland malignancies. There is no established first-line treatment, and prognosis is poor compared to conventional breast cancer. We previously demonstrated the efficacy of cisplatinum and eribulin in a patient-derived orthotopic xenograft (PDOX) mouse model of primary breast osteosarcoma. However, these drugs show significant clinical toxicity. All cancers are addicted to methionine (Hoffman effect). In the present study, we determined whether methionine restriction with oral recombinant methioninase (o-rMETase) would lower the effective dose of cisplatinum in a PDOX model of primary osteosarcoma of the mammary gland, thereby reducing its toxicity. Materials and Methods: Mouse PDOX models of primary osteosarcoma of the breast were randomized into the following groups: control; cisplatinum (weekly at 3 or 6 mg/kg); twice-daily o-rMETase; or o-rMETase combined with 3 mg/kg cisplatinum, with treatment for 2 weeks. Results: Cisplatinum at 6 mg/kg significantly inhibited breast osteo-sarcoma growth compared with the untreated control and mice treated with 3 mg/kg cisplatinum (p=0.01 and 0.009, respectively). There was no significant difference in tumor growth between mice treated with cisplatinum at 3 mg/kg and the control (p=0.16). Combination therapy with cisplatinum at 3 mg/kg and twice daily o-rMETase regressed the osteosarcoma of the mammary gland (p=0.009), similar to the inhibition by cisplatinum at 6 mg/kg alone. Cisplatinum at 6 mg/kg caused a significant loss of mouse body weight, compared to the control (p=0.02). There was no significant body-weight loss with the combination therapy of o-rMETase and cisplatinum at 3 mg/kg, compared to the untreated control. Conclusion: o-rMETase halved the effective dose of cisplatinum, thereby eliminating cisplatinum toxicity, demonstrating a future clinical strategy for therapy of osteosarcoma of the breast.

Invasive lobular carcinoma (ILC) of the breast has a low complete-response rate in the neoadjuvant-chemotherapy setting. The addiction to methionine is a fundamental and ubiquitous characteristic of cancer cells, termed the Hoffman effect. We have previously targeted methionine addiction of breast cancer with recombinant methioninase (rMETase) using patient-derived orthotopic xenograft (PDOX) models. The aim of the present study was to determine the efficacy of methionine restriction with rMETase and a low-methionine diet combined with first-line neo-adjuvant chemotherapy, in a patient with metastatic ILC of the breast.A 62-year-old female was diagnosed with metastatic ipsilateral axillary-lymph-node recurrence of breast ILC 3 years after mastectomy. The patient underwent [Combination therapy of doxorubicin and cyclophosphamide followed by docetaxel combined with methionine restriction led to a remarkable complete response that is expected in fewer than 10% of patients with ILC of the breast treated with neo-adjuvant chemotherapy alone. The present results suggest that methionine restriction in combination with doxorubicin and cyclophosphamide followed by docetaxel may be effective, after METPET has demonstrated the presence of methionine-addicted axillary-lymph-node metastases in ILC of the breast.

Background/aim Methionine addiction is a general and fundamental hallmark of cancer due to the excess use of methionine for transmethylation reactions, termed the "Hoffman Effect". Methionine addiction has been shown to be a highly-effective target for cancer therapy by methionine restriction with oral recombinant methioninase (o-rMETase) in preclinical studies, including patient- derived orthotopic xenograft (PDOX) mouse models of cancer. A clinical study of o-rMETase as a supplement showed a 70% reduction of PSA levels in a patient with bone-metastatic prostate cancer. Materials and methods In the present study, two advanced prostate-cancer patients took o-rMETase as a supplement for approximately one month. Results One of the patients taking o-rMETase showed a 38% reduction of PSA levels and the second patient showed a 20% PSA reduction. Conclusion o-rMETase shows promise for treating patients with advanced prostate cancer.

Advanced ovarian clear cell carcinoma (OCCC) is a recalcitrant disease, often resistant to the first-line platinum-based therapy. Using a novel patient-derived orthotopic xenograft (PDOX) nude-mouse model of OCCC, we tested whether oral-recombinant methioninase (o-rMETase) could enhance the efficacy of paclitaxel (PTX). The OCCC PDOX model was established and passaged in nude mice. The OCCC PDOX models were randomized into 5 groups. G1: untreated control; G2: paclitaxel (PTX) (20 mg/kg, intraperitoneal (i.p.) injection, weekly); G3: o-rMETase (100 units, oral, daily); G4: PTX (20 mg/kg, i.p. injection, weekly) + carboplatinum (CBDCA) (40 mg/kg, i.p. injection weekly); G5: PTX (20 mg/kg, i.p. injection, weekly) + o-rMETase (100 units, oral, daily). The treatment period was 2 weeks. The combination of PTX and o-rMETase arrested OCCC tumor growth (relative tumor volume: 1.09 ± 0.63 (mean ± SD)) compared with the untreated control (relative tumor volume: 3.92 ± 1.04 (mean ± SD)) (p

Background/aim Advanced prostate cancer is a recalcitrant disease with very limited treatment options. Our laboratory discovered methionine addiction, presumably a characteristic of all cancer types, including prostate cancer, which can be targeted by methionine restriction (MR), through treatment with oral recombinant methioninase (o-rMETase). Patients and methods o-rMETase was produced by fermentation of recombinant E. coli containing the Pseudomonas putida methioninase gene, and purified by column chromatography. An advanced prostate cancer patient received o-rMETase as a supplement, 500 units per day, divided into two oral doses of 250 units each. Results Before treatment, the patient had a rapid rise in PSA levels, from 39 to 56 ng/ml, within 6 weeks. At the 15th week of o-rMETase administration, the PSA levels stabilized at 62 ng/ml. No overt side effects were observed. Conclusion o-rMETase single treatment can be beneficial for advanced prostate cancer patients.

Background/aim Dedifferentiated liposarcoma (DDLPS) is associated with a poor survival rate even with multi-modality treatment. In the present study, we evaluated the efficacy of recombinant methioninase (rMETase) combined with tumor-targeting Salmonella typhimurium (S. typhimurium) A1-R against a doxorubicin-resistant DDLPS in a patient-derived orthotopic xenograft (PDOX) mouse model. Materials and methods A recurrent high-grade DDLPS from the right retroperitoneum of a patient was grown orthotopically in the retroperitoneum of nude mice to establish a PDOX model. The PDOX models were randomly divided into the following groups: Control, no treatment; doxorubicin monotherapy; rMETase monotherapy; S. typhimurium A1-R monotherapy; S. typhimurium A1-R and rMETase combination therapy. Tumor length and width were measured before and after treatment. Results On day 14 after treatment, all treatments significantly inhibited DDLPS PDOX tumor growth compared to the untreated control except for doxorubicin monotherapy. rMETase combined with S. typhimurium A1-R was significantly more effective and regressed tumor volume compared to either rMETase or S. typhimurium A1-R alone. The relative body weight did not significantly differ between days 0 and 14 for individual groups. Conclusion The combination of rMETase and S. typhimurium A1-R has important clinical potential for this recalcitrant sarcoma.

Background/aim obesity is a world-wide recalcitrant problem leading to many diseases. Dietary methionine restriction (MR) has been shown to prevent obesity, but it is an onerous regimen. The present study aimed to determine the effects of oral recombinant methionase (o-rMETase), on preventing obesity in mice on a high-fat diet. Materials and methods Male C57BL/6J mice in the control group were fed a control diet (CD) (+6.5% fat) for 25 days, and others were fed a high-fat (HF) diet (+34.3% fat) for 25 days. Then, the mice were divided into three dietary groups: 1) HF + phosphate buffered saline (PBS) group; 2) HF + o-rMETase group; and 3) untreated non-HF group. Results The mice on the CD increased in body weight by 14% during experimental period of 25 days; in contrast the mice in the HF+PBS group increased by 33%; however, the mice on the HF+o-rMETase group increased only by 14% (p=0.02, HF+PBS vs HF+o-rMETase). Conclusion The HF+ o-rMETase group had the same weight increase as untreated mice on a normal fat diet, demonstrating the potential for o-rMETase to eliminate the need for dieting to maintain normal body weight.

Background/Aim: Pazopanib (PAZ) can inhibit tumor progression, but whether PAZ inhibits lymph node metastasis and lymphangiogenesis in colorectal cancer is still unknown. The aim of the present study was to determine the efficacy of PAZ on tumor growth, lymph node metastasis and lymphangiogenesis in an orthotopic nude mouse model in colorectal cancer. Materials and Methods: CT-26-green fluorescence protein (GFP)-expressing mouse colon cancer cells were injected into nude mice to establish a subcutaneous colorectal cancer model and were treated with saline and PAZ. Additionals subcutaneous tumors were harvested and cut into 5 mm(3 )fragments, then tumor fragments were implanted orthotopically in the cecum to establish an orthotopic colorectal-cancer nude mouse model. Orthotopic mice were randomized into two groups for the treatment with saline and PAZ, respectively. Tumor width, length and mouse body weight was measured twice a week. The Fluor Vivo imaging system was used to image the GFP. Hematoxylin & eosin staining and immunohistochemical staining was used for histological analysis. Results: PAZ inhibited the growth of subcutaneous colorectal cancer, as wells as orthotopic transplanted colorectal cancer tumors. PAZ suppressed lymph node metastasis and lymphangiogenesis in the orthotopic colon cancer model. No significant changes were observed in the body weight between the control and the mice treated with PAZ. Conclusion: PAZ can inhibit the growth of colorectal cancer and inhibit lymph node metastasis and lymphangiogenesis in orthotopic colon cancer nude mouse models.

Background/aim We have recently shown that oral recombinant methionase (o-rMETase) prevents obesity and diabetes onset in mice on a high-fat (HF) diet. The present study aimed to determine if o-rMETase can inhibit the onset of nonalcoholic fatty liver disease (NAFLD) onset in mice on a high-fat diet. Materials and methods Male C57BL/6J mice in the control group were fed a normal-fat diet (NFD) (+6.5% fat), and other mice were fed a high-fat (HF) diet (+34.3% fat). Then, the mice on the HF diet were divided into two dietary groups: i) HF+phosphate buffered saline (PBS) group, and ii) HF+o-rMETase group. Result The fatty change score in the livers of mice treated with HF+PBS increased to an average of 2.6 during the experimental period of 8 weeks. In contrast, the fatty change in the livers of mice on the HF+o-rMETase group had an average score of 0.92 (p=0.04, HF+PBS vs HF+o-rMETase). Conclusion o-rMETase inhibited the onset of NAFLD as well as prevented obesity and the onset of diabetes on a high-fat diet, offering a possibility of a new paradigm to prevent liver cirrhosis or liver cancer via NAFLD.

The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other diseases, it is thought that targeting the biology of the SARS-CoV-2 virus, which causes Covid-19, can result in an effective therapeutic treatment. The coronavirus RNA cap structure is methylated by two viral methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM). The proper methylation of the virus depends on the level of methionine in the host to form SAM. Herein, we propose to restrict methionine availability by treating the patient with oral recombinant methioninase, aiming to treat Covid-19. By restricting methionine we not only interdict viral replication, which depends on the viral RNA cap methyaltion, but also inhibit the proliferation of the infected cells, which have an increased requirement for methionine. Most importantly, the virally-induced T-cell- and macrophage-mediated cytokine storm, which seems to be a significant cause for Covid-19 deaths, can also be inhibited by restricting methionine, since T-cell and macrophrage activation greatly increases the methionine requirement for these cells. The evidence reviewed here suggests that oral recombinant methioninase could be a promising treatment for coronavirus patients.

Pancreatic cancer is one of the most recalcitrant cancers, and more effective therapy is needed. Pre-clinical studies have shown that patient-derived orthotopic xenograft (PDOX) mouse models of pancreatic cancer are effectively treated with oral recombinant methioninase (o-rMETase).A 62-year-old woman diagnosed with stage IV pancreatic cancer was treated with the combination of 5-fluorouracil/leucovorin, irinotecan, and oxaliplatinum (FOLFIRINOX) every two weeks and o-rMETase twice a day as a supplement. The patient was also on a low-methionine diet. Disease progression was monitored by CA19-9 and computed tomography. The patient initially responded to FOLFIRINOX, shown by a great reduction in CA19-9 levels, with tumor shrinkage shown by computed tomography. The patient began taking o-rMETase and went on a low-methionine diet one year after diagnosis which she has maintained without side effects for 7 months. The patient's CA19-9 level and tumor size remain stable 19 months after diagnosis. The patient is alive and has maintained a high performance status. Historical data show that less than 5% of stage IV pancreatic-cancer patients on FOLFIRINOX have stable disease 1.5 years after diagnosis.The combination of o-rMETase and FOLFIRINOX may be synergistic in stage IV pancreatic cancer.

The aim of this study was to determine the efficacy of oral recombinant methioninase (o-rMETase) on a model of colon cancer growing on the peritoneal surface using a patients-derived orthotopic xenograft (PDOX) nude mouse model. Forty PDOX mouse models with colon cancer growing on the peritoneum were divided into 4 groups of 10 mice each by measuring the tumor size and fluorescence intensity: untreated control; 5-fluorouracil (5-FU) (50 mg/kg, once a week for two weeks, ip) and oxaliplatinum (OXA) (6 mg/kg, once a week for two weeks, ip); o-rMETase (100 units/day, oral 14 consecutive days); combination 5-FU + OXA and o-rMETase. All treatments inhibited tumor growth compared to the untreated control. The combination of 5-FU + OXA plus o-rMETase was significantly more efficacious than the control and each drug alone and was the only treatment that caused tumor regression. The present study is the first demonstrating the efficacy of o-rMETase combination therapy on a PDOX model of peritoneal colon cancer, suggesting potential clinical development of o-rMETase in a recalcitrant cancer.

Background/aim The aim of the present study was to determine the efficacy of trabectedin combined with FOLFIRI (irinotecan, leucovorin and 5-fluorouracil) on a colorectal cancer (CRC) patient-derived orthotopic xenograft (iPDOX) mouse model. Materials and methods A CRC tumor from a patient previously established in nude mice was implanted subcutaneously in transgenic green fluorescence protein (GFP)-expressing nude mice in order to label the tumor stromal cells with GFP. Mice were randomized into four groups: Group 1, untreated control; group 2, FOLFIRI; group 3, trabectedin alone; group 4, trabectedin plus FOLFIRI. Tumor width, length, and mouse body weight was measured twice every week. Results All three treatment groups showed inhibited tumor growth compared to the untreated control group. Only the combination of FOLFIRI and trabectedin arrested tumor growth. No significant changes was observed in body weight in any group. Conclusion These findings suggest that the combination of trabectedin plus FOLFIRI has clinical potential for patients with CRC.

Background/Aim: Methionine addiction is a fundamental and general hallmark of cancer cells, which require exogenous methionine, despite large amounts of methionine synthesized endogenously. 5-Methylthioadenosine phospho-rylase (MTAP) plays a principal role as an enzyme in the methionine-salvage pathway, which produces methionine and adenine from methylthioadenosine and is deleted in 27.5% to 37.5% of osteosarcoma patients. Materials and Methods: Human osteosarcoma cell lines U2OS, SaOS2, MNNG/HOS (HOS) and 143B, were used. The MTAP gene was knocked out in U2OS with CRISPR/Cas9. 143B and HOS have an MTAP deletion and SaOS2 is positive for MTAP. MTAP was determined by western blotting. The four cell lines were compared for sensitivity to recombinant methioninase (rMETase). Results: MTAP-deleted osteosarcoma cell lines MNNG/HOS and 143B were significantly more sensitive to rMETase than MTAP-positive osteosarcoma cell lines U2OS and SaOS2. In addition, MTAP knock-out U2OS cells were more sensitive to rMETase than the parental MTAP-positive U2OS cells. Conclusion: The present results demonstrated that the absence of MTAP sensitizes osteosarcoma cells to methionine restriction by rMETase, a promising clinical strategy.

Methionine addiction is a fundamental and general hallmark of cancer cells, which require exogenous methionine, despite their ability to synthesize normal amounts of methionine from homocysteine. In contrast, methionine-independent normal cells do not require exogenous methionine in the presence of a methionine precursor. The methionine addiction of cancer cells is due to excess transmethylation reactions. We have previously shown that histone H3 lysine marks are over-methylated in cancer cells and the over-methylation is unstable when the cancer cells are restricted of methionine. In the present study, we show that methionine-addicted osteosarcoma cells are sensitive to both methotrexate (MTX) and recombinant methioninase (rMETase), but they affect histone H3 lysine-methylation in the opposite direction. Concentrations of MTX and rMETase, which inhibit osteosarcoma cells viability to 20%, had opposing effects on the status of histone methylation of H3K9me3 and H3K27me3. rMETase significantly decreased the amount of H3K9me3 and H3K27me3. In contrast, MTX significantly increased the amount of H3K9me and H3K27me3. The results suggest that increase or decrease in these methylated histone lysine marks is associated with proliferation arrest of methionine-addicted osteosarcoma., Highlights • Osteosarcoma cells are sensitive to both methotrexate and recombinant methioninase. • MTX increased the amount of H3K9me and H3K27me3. • RMETase decreased the amount of H3K9me3 and H3K27me3. • Increase/decrease in H3K9me3 and H3K27me3 is associated with proliferation arrest.

Methionine addiction, found in all types of cancer investigated, is because of the overuse of methionine by cancer cells for excess transmethylation reactions. In the present study, we compared the histone H3 lysine-methylation status and degree of malignancy between methionine-addicted cancer cells and their isogenic methionine-independent revertants, selected by their growth in low concentration of methionine. The methionine-independent revertans can grow on low levels of methionine or independently of exogenous methionine using methionine precursors, as do normal cells. In the methionine-independent revertants, the excess levels of trimethylated histone H3 lysine marks found in the methionine-addicted parental cancer cells were reduced or lost, and their tumorigenicity and experimental metastatic potential in nude mice were also highly reduced. Methionine addiction of cancer is linked with malignancy and hypermethylation of histone H3 lysines. The results of the present study thus provide a unique framework to further understand a fundamental basis of malignancy.

Background/Aim: Methionine addiction is a fundamental and general hallmark of cancer, termed the Hoffman effect. Methionine addiction is due to excessive use of and dependence on methionine by cancer cells. In the present report, we correlated the extent of methionine addiction and degree of malignancy with the amount and stability of methylated histone H3 lysine marks. Materials and Methods: We established low- and high-malignancy variants from a parental human pancreatic-cancer cell line and compared their sensitivity to methionine restriction and histone H3 lysine methylation status. Results: A low-malignancy, low-methionine-addiction revertant of the parental pancreatic-cancer cell line had less methylated H3K9me3 and was less sensitive to methionine restriction effected by recombinant methioninase (rMETase) than the parental cell line. A high-malignancy variant of the pancreatic cancer cell line had increased methylated H3K9me3 and was more sensitive to methionine restriction by rMETase with regard to inhibition of proliferation and to instability of histone H3 lysine methylation than the parental cell line. Orthotopic malignancy in nude mice was reduced in the low-methionine-addiction revertant and greater in the high-malignancy variant than in the parental cell line. Conclusion: The present study indicates that the degree of malignancy is linked to the extent of methionine addiction and the level and instability of trimethylation of histone H3, suggesting these phenomena are linked as a fundamental basis of oncogenic transformation.