Preparation of Internalizing RGD-Modified Recombinant Methioninase Exosome Active Targeting Vector and Antitumor Effect Evaluation.

BACKGROUND/AIMS: Targeted drug delivery vehicles with low immunogenicity and toxicity are needed for cancer therapy. Here, we prepare an active targeting drug carrier of low immunogenicity and toxicity for targeted therapy. METHODS: Immature dendritic cells (imDCs) from BALB/c mice were used as donor cells of exosomes (Exos) that were transfected with the plasmids expressing fusion proteins of a tumor-targeting peptide known as internalizing RGD (iRGD) to construct a type of tumor-targeting iRGD-Exos and observe the interaction between these iRGD-Exos. Also, recombinant methioninase (rMETase) was loaded into the iRGD-Exos by electroporation to construct iRGD-Exos-rMETase and to assess the tumor-targeting function of the iRGD-Exos-rMETase. Finally, 30 BALB/c were randomly divided into five groups (n = 6), to observe tumor growth in vivo. RESULTS: The iRGD-Exos-rMETase was 99.58 nm in diameter and presented a unique "goblet" structure under transmission electron microscopy (TEM), with the encapsulation efficiency (EE) of 19.05%. iRGD-Exos-rMETase group has the strongest tumor suppressive effect. Compared to the iRGD-Exos-rMETase group, rMETase group and the blank-Exos-rMETase group were less effective, while the PBS group and the iRGD-Exos group showed no inhibitory effect on tumor growth. After treatment, the iRGD-Exos-rMETase group had gastric tumors significantly smaller and lighter than the other groups (P < 0.05). CONCLUSION: The iRGD-Exos-rMETase is an effective antitumor therapy that delivers rMETase to tumor tissue using the iRGD-Exos. With its favorable inhibitory effect and tumor-targeting function, the iRGD-Exos-rMETase shows excellent potential value and exciting prospects in clinical applications.

Combination of oral recombinant methioninase and decitabine arrests a chemotherapy-resistant undifferentiated soft-tissue sarcoma patient-derived orthotopic xenograft mouse model.

Cancer cells are methionine (MET) and methylation addicted and are highly sensitive to MET restriction. The present study determined the efficacy of oral-recombinant methioninase (o-rMETase) and the DNA methylation inhibitor, decitabine (DAC) on restricting MET in an undifferentiated-soft tissue sarcoma (USTS) patient-derived orthotopic xenograft (PDOX) nude-mouse model. The USTS PDOX models were randomized into five treatment groups of six mice: Control; doxorubicin (DOX) alone; DAC alone; o-rMETase alone; and o-rMETase-DAC combination. Tumor size and body weight were measured during the 14 days of treatment. Tumor growth was arrested only in the o-rMETase-DAC condition. Tumors treated with the o-rMETase-DAC combination exhibited tumor necrosis with degenerative changes. This study demonstrates that the o-rMETase-DAC combination could arrest the USTS PDOX tumor suggesting clinical promise.

Growth of doxorubicin-resistant undifferentiated spindle-cell sarcoma PDOX is arrested by metabolic targeting with recombinant methioninase.

Undifferentiated spindle-cell sarcoma (USCS) is a recalcitrant -cancer in need of individualized therapy. A high-grade USCS from a striated muscle of a patient was grown orthotopically in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. In a previous study, we evaluated the efficacy of standard first-line chemotherapy of doxorubicin (DOX), gemcitabine (GEM) combined with docetaxel (DOC), compared to pazopanib (PAZ), a multi-targeting tyrosine-kinase inhibitor, in an USCS PDOX model. In the present study, mice-bearing the USCS PDOX tumors were randomized into the following groups when tumor volume reached 100 mm3 : G1, untreated control without treatment; G2, DOX (3 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, L-methionine α-deamino-γ-mercaptomethane lyase (recombinant methioninase [rMETase]) (100 U/mouse, i.p., daily, for 2 weeks). Tumor size and body weight were measured with calipers and a digital balance twice a week. The methionine level of supernatants derived from sonicated tumors was also measured. rMETase inhibited tumor growth, measured by tumor volume, compared to untreated controls and the DOX-treated group on day 14 after initiation of treatment: control (G1): 347.6 ± 88 mm3 ; DOX (G2): 329.5 ± 79 mm3 , P = 0.670; rMETase (G3): 162.6 ± 51 mm3 , P = 0.0003. The mouse body weight of the treated mice was not significantly different from the untreated controls. Tumor L-methionine levels were reduced after the rMETase-treatment compared to untreated control and pre-rMETase treatment. We previously reported efficacy of rMETase against Ewing's sarcoma and melanoma in a PDOX models. These studies suggest clinical development of rMETase, especially in recalcitrant cancers such as sarcoma.

Combination therapy of tumor-targeting Salmonella typhimurium A1-R and oral recombinant methioninase regresses a BRAF-V600E-negative melanoma.

Melanoma is a recalcitrant cancer. To improve and individualize treatment for this disease, we previously developed a patient-derived orthotopic xenograft (PDOX) model for melanoma. We previously reported the individual efficacy of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and recombinant methioninase (rMETase) for melanoma in the PDOX models of this disease. In the present study, we evaluated the efficacy of the combination of S. typhimurium A1-R with orally-administered rMETase (o-rMETase) for BRAF-V600E-negative melanoma in a PDOX model. Three weeks after implantation, 60 PDOX mouse models were randomized into six groups of 10 mice each: untreated control, temozolomide (TEM); o-rMETase; S. typhimurium A1-R; TEM + rMETase, S. typhimurium A1-R + rMETase. All treatments inhibited tumor growth compared to untreated control (TEM: p < 0.0001, rMETase: p < 0.0001, S. typhimurium A1-R: p < 0.0001, TEM + rMETase: p < 0.0001, S. typhimurium A1-R + rMETase: p < 0.0001). The most effective was the combination of S. typhimurium A1-R + o-rMETase which regressed this melanoma PDOX, thereby indicating a new paradigm for treatment of metastatic melanoma.

Expression of PD-L1 Is Increased by Methionine Restriction Using Recombinant Methioninase in Human Colorectal Cancer Cells.

BACKGROUND/AIM: It has been recently demonstrated that a methionine-restricted diet increases the response to immune checkpoint inhibitors (ICIs) via an increase in PD-L1 in a syngeneic mouse colorectal-cancer model. Our laboratory has developed recombinant methioninase (rMETase) to restrict methionine. The aim of the present study was to determine if rMETase can increase PD-L1 expression in a human colorectal cancer cell line in vitro. MATERIALS AND METHODS: We evaluated the half-maximal inhibitory concentration (IC50) value of rMETase on HCT-116 human colorectal cancer cells. HCT-116 cells were treated with rMETase at the IC50 Western immunoblotting was used to compare PD-L1 expression in HCT-116 cells treated with and without rMETase. RESULTS: The IC50 value of rMETase on HCT-116 was 0.79 U/ml. Methionine restriction using rMETase increased PD-L1 expression compared to the untreated control (p<0.05). CONCLUSION: Methionine restriction with rMETase up-regulates PD-L1 expression in human colorectal cancer cells and the combination of rMETase and ICIs may have the potential to improve immunotherapy in human colorectal cancer.

The Combination of Methionine Restriction and Docetaxel Synergistically Arrests Androgen-independent Prostate Cancer But Not Normal Cells.

Background/Aim: Androgen-independent prostate cancer (AIPC) is resistant to androgen-depletion therapy and is a recalcitrant disease. Docetaxel is the first-line treatment for AIPC, but has limited efficacy and severe side-effects. All cancers are methionine-addicted, which is termed the Hoffman effect. Recombinant methioninase (rMETase) targets methionine addiction. The purpose of the present study was to determine if the combination of docetaxel and rMETase is effective for AIPC. Materials and Methods: The half-maximal inhibitory concentrations (IC50) of docetaxel and rMETase alone were determined for the human AIPC cell line PC-3 and Hs27 normal human fibroblasts in vitro. The synergistic efficacy for PC-3 and Hs27 using the combination of docetaxel and rMETase at their IC50s for PC-3 was determined. Results: The IC50 of docetaxel for PC-3 and for Hs27 was 0.72 nM and 0.94 nM, respectively. The IC50 of rMETase for PC-3 and for Hs27 was 0.67 U/ml and 0.76 U/ml, respectively. The combination of docetaxel and rMETase was synergistic for PC-3 but not Hs27 cells. Conclusion: The combination of a relatively low concentration of docetaxel and rMETase was synergistic and effective for AIPC. The present results also suggest that the effective concentration of docetaxel can be reduced by using rMETase, which may reduce toxicity. The present results also suggest the future clinical potential of the combination of docetaxel and rMETase for AIPC.

Old-age-induced obesity reversed by a methionine-deficient diet or oral administration of recombinant methioninase-producing Escherichia coli in C57BL/6 mice.

Obesity increases with aging. Methionine restriction affects lipid metabolism and can prevent obesity in mice. In the present study we observed C57BL/6 mice to double their body weight from 4 to 48 weeks of age and become obese. We evaluated the efficacy of oral administration of recombinant-methioninase (rMETase)-producing E. coli (E. coli JM109-rMETase) or a methionine-deficient diet to reverse old-age-induced obesity in C57BL/6 mice. Fifteen C57BL/6 male mice aged 12-18 months with old-age-induced obesity were divided into three groups. Group 1 was given a normal diet supplemented with non-recombinant E. coli JM109 cells orally by gavage twice daily; Group 2 was given a normal diet supplemented with recombinant E. coli JM109-rMETase cells by gavage twice daily; and Group 3 was given a methionine-deficient diet without treatment. The administration of E. coli JM109-rMETase or a methionine-deficient diet reduced the blood methionine level and reversed old-age-induced obesity with significant weight loss by 14 days. There was a negative correlation between methionine levels and negative body weight change. Although the degree of efficacy was higher in the methionine-deficient diet group than in the E. coli JM109-rMETase group, the present findings suggested that oral administration of E. coli JM109-rMETase, as well as a methionine-deficient diet, are effective in reversing old-age-induced obesity. In conclusion, the present study provides evidence that restricting methionine by either a low-methionine diet or E. coli JM109-rMETase has clinical potential to treat old-age-induced obesity.

Synergy of Combining Methionine Restriction and Chemotherapy: The Disruptive Next Generation of Cancer Treatment.

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.

Colon-cancer liver metastasis is effectively targeted by recombinant methioninase (rMETase) in an orthotopic mouse model.

BACKGROUND: Colorectal cancer liver metastasis (CCLM) is the most frequent cause of death of colorectal cancer. Development of novel new effective therapy is needed for CCLM patients to improve outcome. The aim of the present study was to investigate the efficacy of recombinant methioninase (rMETase) on a CCLM orthotopic mouse model of liver metastasis established using the human colon cancer cell line HT29 expressing red fluorescent protein (RFP). MATERIALS AND METHODS: Orthotopic CCLM nude mouse models were randomized into two groups: control group (n = 6, PBS 200 µl, i.p., daily); rMETase group (n = 6, 100 units/200 µl, i.p., daily). Tumor volume was measured on day 0 and day 15. Body weight was measured twice a week. All mice were sacrificed on day 15. RESULTS: rMETase significantly inhibited the increase of the liver metastasis as determined by RFP fluorescence area and intensity (p = 0.016 and 0.015, respectively). There was no significant difference of body weight between either group on any day. CONCLUSIONS: The present study suggests that rMETase has future potential therapy for CCLM in the clinic.

Recombinant-methioninase-producing Escherichia coli Instilled in the Microbiome Inhibits Triple-negative Breast Cancer in an Orthotopic Cell-line Mouse Model.

Background/Aim: Methionine restriction by diet and recombinant methioninase (rMETase) are effective for cancer therapy by themselves or combined with chemotherapy drugs. We previously showed that oral administration of rMETase-producing Escherichia coli JM109 (E. coli JM109-rMETase) can be installed in the mouse microbiome and inhibit colon-cancer growth in a syngeneic mouse model. In the present report, we investigated the efficacy of oral administration of E. coli JM109-rMETase in an orthotopic triple-negative breast cancer (TNBC) cell-line mouse model. Materials and Methods: First, we established orthotopic 4T1 mouse triple-negative breast cancer on an abdominal mammary gland in female athymic nu/nu nude mice aged 4-6 weeks. After tumor growth, 15 mice were divided into three groups of 5. Group 1 was administered phosphate-buffered saline (PBS) orally by gavage twice daily as a control; Group 2 was administered non-recombinant E. coli JM109 competent cells orally by gavage twice daily as a control; Group 3 was administered E. coli JM109-rMETase cells by gavage twice daily for two weeks. Tumor size was measured with calipers twice per week. On day 15, blood methionine level was examined using an HPLC method. Results: Oral administration of E. coli JM109-rMETase inhibited 4T1 TNBC growth significantly compared to the PBS and E. coli JM109 control groups. On day 15, the blood methionine level was significantly lower in the mice administered E. coli JM109-rMETase than in the PBS control. Conclusion: E. coli JM109-rMETase lowered blood methionine levels and inhibited TNBC growth in an orthotopic cell-line mouse model, suggesting future clinical potential against a highly recalcitrant cancer.

Rapid Reduction of CEA and Stable Metastasis in an NRAS-mutant Rectal-Cancer Patient Treated With FOLFIRI and Bevacizumab Combined With Oral Recombinant Methioninase and a Low-Methionine Diet Upon Metastatic Recurrence After FOLFIRI and Bevacizumab Treatment Alone.

BACKGROUND/AIM: The choice of chemotherapy agents for RAS-mutant colorectal cancer is limited, and prognosis is poor compared to RAS-wild-type colorectal cancer. The purpose of the present study was to evaluate the effectiveness of methionine restriction combined with chemotherapy in a patient with NRAS-mutant rectal cancer. PATIENTS AND METHODS: A 59-year-old female was diagnosed with lung-metastatic recurrence of NRAS-mutant rectal cancer two and a half years after resection of the primary tumor. She started chemotherapy, which consisted of fluorouracil, irinotecan (FOLFIRI), and bevacizumab, in October 2020. Eight months later, stereotactic body radiation therapy (SBRT) was performed to treat the lung metastases. She stopped chemotherapy at this point and had blood tests and computed tomography (CT) scans regularly. Her CEA level increased to 139.91 ng/ml and her lung metastasis became larger by September 2022. Therefore, she was reintroduced to FOLFIRI and bevacizumab in October 2022, and also started a low-methionine diet and oral recombinant methioninase (o-rMETase) as a supplement. RESULTS: After starting the combination therapy with o-rMETase, a low-methionine diet, FOLFIRI, and bevacizumab, blood CEA levels very rapidly decreased and were almost within the normal limits five months later. CT findings showed the lung metastasis did not grow. CONCLUSION: Methionine restriction comprising o-rMETase and a low-methionine diet combined with first-line chemotherapy was effective in a patient with NRAS-mutant rectal cancer in which metastasis had re-occurred after first-line chemotherapy alone.

Deletion of MTAP Highly Sensitizes Osteosarcoma Cells to Methionine Restriction With Recombinant Methioninase.

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 phosphorylase (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.

Oral Installation of Recombinant Methioninase-producing Escherichia coli into the Microbiome Inhibits Colon-cancer Growth in a Syngeneic Mouse Model.

BACKGROUND/AIM: All cancer types so far tested are methionine-addicted. Targeting the methionine addiction of cancer with recombinant methioninase (rMETase) has shown great progress in vitro, in mouse models, and in the clinic. However, administration of rMETase requires multiple doses per day. In the present study, we determined if rMETase-producing Escherichia coli JM109 (E. coli JM109-rMETase) might be an effective anticancer agent when installed into the microbiome. MATERIALS AND METHODS: E. coli JM109-rMETase was administered to a syngeneic model of MC38 colon cancer growing subcutaneously in C57BL/6 mice. JM109-rMETase was administered orally by gavage to the mice twice per day. Tumor size was measured with calipers. RESULTS: The administration of E. coli JM109-rMETase twice a day significantly inhibited MC38 colon-cancer growth. E. coli JM109-rMETase was found in the stool of treated mice, indicating it had entered the microbiome. CONCLUSION: The present study indicates the potential of microbiome-based treatment of cancer targeting methionine addiction.

Stage IV Pancreatic Cancer Patient Treated With FOLFIRINOX Combined With Oral Methioninase: A Highly-Rare Case With Long-term Stable Disease.

BACKGROUND: 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). CASE REPORT: 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. CONCLUSION: The combination of o-rMETase and FOLFIRINOX may be synergistic in stage IV pancreatic cancer.

Long-term Stable Disease in a Rectal-cancer Patient Treated by Methionine Restriction With Oral Recombinant Methioninase and a Low-methionine Diet.

BACKGROUND/AIM: 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. PATIENT AND METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Elimination of Axillary-Lymph-Node Metastases in a Patient With Invasive Lobular Breast Cancer Treated by First-line Neo-adjuvant Chemotherapy Combined With Methionine Restriction.

BACKGROUND/AIM: 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. CASE REPORT: A 62-year-old female was diagnosed with metastatic ipsilateral axillary-lymph-node recurrence of breast ILC 3 years after mastectomy. The patient underwent [11C]-methionine positron-emission tomography (METPET) which showed extensive methionine accumulation in the ipsilateral axillary lymph nodes, indicating the presence of cancer cells. The patient received standard neo-adjuvant chemotherapy, which consisted of 3 months of doxorubicin and cyclophosphamide followed by 3 months of docetaxel from March 2022. The patient also went on a low-methionine diet and daily oral rMETase as a supplement every 6 hours concurrently with six months chemotherapy. The patient's blood carcinoembryonic antigen (CEA) level decreased gradually, and computed tomography findings showed loss of axillary lymph-node metastases in the first 3 months of neo-adjuvant chemotherapy with doxorubicin and cyclophosphamide combined with rMETase and a low-methionine diet. A complete response was demonstrated by METPET at 6 months, at conclusion of docetaxel chemotherapy. CONCLUSION: 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.

Efficacy of Oral Recombinant Methioninase and Eribulin on a PDOX Model of Triple-negative Breast Cancer (TNBC) Liver Metastasis.

BACKGROUND/AIM: The aim of the present study was to identify effective drugs for a highly-aggressive liver-metastasis of triple-negative breast cancer (TNBC) in a patient-derived orthotopic xenograft (PDOX) mouse model. Drugs tested were oral recombinant methioninase (o-rMETase), low-dose eribulin and their combination. MATERIALS AND METHODS: Patient-derived TNBC was implanted in the liver of nude mice by surgical hepatic implantation. Two weeks after transplantation, 32 mice were randomized (n=8 per group) into a phosphate-buffered saline vehicle-control group; o-rMETase-treatment group (100 units, o-rMETase, oral, daily for 2 weeks); eribulin-treatment group (0.05 mg/kg intraperitoneally once per week for 2 weeks); or combination-treatment group (100 units r-METase, oral, daily for 2 weeks + 0.05 mg/kg eribulin intraperitoneally once per week for 2 weeks). RESULTS: After 2 weeks, the three treatment groups exhibited significantly-inhibited TNBC growth in the liver compared to the vehicle-control group (p≤0.05). CONCLUSION: o-rMETase and low-dose eribulin monotherapy and their combination were efficacious against the highly-aggressive TNBC PDOX growing in the liver. The TNBC PDOX model can be used to identify highly-effective drugs for therapy of TNBC with liver metastasis.

Histone H3 lysine-trimethylation markers are decreased by recombinant methioninase and increased by methotrexate at concentrations which inhibit methionine-addicted osteosarcoma cell proliferation.

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.

Oral recombinant methioninase combined with paclitaxel arrests recalcitrant ovarian clear cell carcinoma growth in a patient-derived orthotopic xenograft (PDOX) nude-mouse model.

PURPOSE: 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). METHODS: 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. RESULTS: 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 < 0.0001). There was no significant difference in relative tumor volume between PTX plus o-rMETase and PTX plus CBDCA (relative tumor volume: 1.39 ± 0.37 (mean ± SD)) (p = 0.93). CONCLUSION: PTX plus o-rMETase arrested the OCCC tumor growth. o-rMETase is readily administered and can greatly enhance first-line therapy of a recalcitrant cancer. The novel and effective treatment strategy in the present report has future clinical potential for patients with OCCC, especially for patients who cannot well tolerate platinum-based therapy.

The combination of oral-recombinant methioninase and azacitidine arrests a chemotherapy-resistant osteosarcoma patient-derived orthotopic xenograft mouse model.

PURPOSE: Cancers are methionine (MET) and methylation addicted, causing them to be highly sensitive to MET restriction. The present study determined the efficacy of restricting MET with oral-recombinant methioninase (o-rMETase) and the DNA methylation inhibitor, azacitidine (AZA) on a chemotherapy-resistant osteosarcoma patient-derived orthotopic xenograft (PDOX) mouse model. METHODS: The osteosarcoma PDOX models were randomized into five treatment groups of six mice: control; doxorubicin (DOX) alone; AZA alone; o-rMETase alone; o-rMETase-AZA combination. Tumor size and body weight were measured during the 14 days of treatment. RESULTS: We found that tumor growth was arrested only by the o-rMETase-AZA combination treatment, as tumors with this treatment exhibited tumor necrosis with degenerative change. CONCLUSION: This study suggests that o-rMETase-AZA combination has clinical potential for patients with chemoresistant osteosarcoma.