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.

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

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.

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.

Head-to-head Comparison of Green Fluorescent Protein (GFP) Imaging With Luciferase-luciferin Imaging In Vivo Using Single-nanometer Laser-excitation Tuning and an Ultra-low-light-detection Camera and Optics Demonstrates the Superiority of GFP.

BACKGROUND/AIM: Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. RESULTS: The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. CONCLUSION: An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.

DNA-Binding Agent Trabectedin Combined With Recombinant Methioninase Is Synergistic to Decrease Fibrosarcoma Cell Viability and Induce Nuclear Fragmentation But Not Synergistic on Normal Fibroblasts.

BACKGROUND/AIM: The alkylating agent trabectedin, which binds the minor groove of DNA, is second-line therapy for soft-tissue sarcoma but has only moderate efficacy. The aim of the present study was to determine the synergistic efficacy of recombinant methioninase (rMETase) and trabectedin on fibrosarcoma cells in vitro, compared with normal fibroblasts. MATERIALS AND METHODS: HT1080 human fibrosarcoma cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and Hs27 normal human fibroblasts, were used. Each cell line was cultured in vitro and divided into four groups: no-treatment control; trabectedin treated; rMETase treated; and trabectedin plus rMETase treated. The dual-color HT1080 cells were used to quantitate nuclear fragmentation in each treatment group. RESULTS: The combination of rMETase and trabectedin was highly synergistic to decrease HT1080 cell viability. In contrast, there was no synergy on Hs27 cells. Moreover, nuclear fragmentation occurred synergistically with the combination of trabectedin and rMETase on dual-color HT1080 cells. CONCLUSION: The combination treatment of trabectedin plus rMETase was highly synergistic on fibrosarcoma cells in vitro suggesting that the combination can improve the outcome of trabectedin alone in future clinical studies. The lack of synergy of rMETase and trabectedin on normal fibroblasts suggests the combination is not toxic to normal cells. Synergy of the two drugs may be due to the high rate of nuclear fragmentation on treated HT1080 cells, and the late-S/G2 cell-cycle block of cancer cells by rMETase, which is a target for trabectedin. The results of the present study suggest the future clinical potential of the combination of rMETase and trabectedin for soft-tissue sarcoma.

Clinical features of patients with carcinoma soft tissue metastases as surgical indications: a retrospective cohort study.

BACKGROUND: Soft-tissue metastasis of carcinoma is rare. In the present study, we investigated the surgical indications and clinical features of patients with soft tissue metastases of carcinoma. METHODS: In this retrospective cohort study, we enrolled 26 patients with soft tissue carcinoma metastasis referred to our department for treatment. Sex, age, location, size, depth, pain due to the tumor, primary origin, serum C-reactive protein (CRP) level, MRI examinations, diagnosis by a previous physician, carcinoma markers from blood, history of carcinoma, other metastases, performance status (PS), and surgical procedures were documented. Associations between variables and surgery were statistically analyzed. RESULTS: The primary cancer origin was found to be the lung (n = 10), kidney (n = 7), esophagus (n = 2), stomach (n = 1), breast (n = 1), liver (n = 1), ureter (n = 1), anus (n = 1), and unknown (n = 2). The mean CRP level of all patients was 2.3 mg/dL. Seven tumors (26.9%) were originally suspected to be soft tissue metastases of carcinoma, while 19 tumors (73.1%) were considered soft tissue sarcomas or inflammatory lesions by the previous treating physician. Twenty patients (76.9%) had other metastases. The PS of the 12 patients (46.2%) was zero. Eleven patients (42.3%) underwent surgery for soft tissue metastases. Diagnosis of soft tissue metastasis by a previous physician and good PS (p < 0.05) were significantly associated with surgery. CONCLUSION: Overall, the present results show that surgical indications for soft tissue metastasis of carcinoma include diagnosis by the referring physician or good PS of the patients.

Extensive Shrinkage and Long-term Stable Disease in a Teenage Female Patient With High-grade Glioma Treated With Temozolomide and Radiation in Combination With Oral Recombinant Methioninase and a Low-methionine Diet.

BACKGROUND/AIM: Gliomas are the most common and recalcitrant malignant primary brain tumors. All cancer types are addicted to methionine, which is a fundamental and general hallmark of cancer known as the Hoffman effect. Particularly glioma cells exhibit methionine addiction. Because of methionine addiction, [11C]-methionine positron emission tomography (MET-PET) is widely used for glioma imaging in clinical practice, which can monitor the extent of methionine addiction. Methionine restriction including recombinant methioninase (rMETase) and a low-methionine diet, has shown high efficacy in preclinical models of gliomas, especially in combination with chemotherapy. The aim of the present study was to determine the efficacy of methionine restriction with oral rMETase (o-rMETase) and a low-methionine diet, combined with radiation and temozolomide (TMZ), on a teenage female patient with high-grade glioma. CASE REPORT: A 16-year-old girl was diagnosed with high-grade glioma. Magnetic resonance imaging (MRI) showed a left temporal-lobe tumor with compression to the left lateral ventricle and narrowing of sulci in the left temporal lobe. After the start of methionine restriction with o-rMETase and a low-methionine diet, along with TMZ combined with radiotherapy, the tumor size shrunk at least 60%, with improvement in the left lateral ventricle and sulci. The patient's condition remains stable for 19 months without severe adverse effects. CONCLUSION: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with radiation and TMZ as first-line chemotherapy, were highly effective in a patient with high-grade glioma.

Development and validation of a nomogram to predict surgical site infection after soft-tissue sarcoma resection.

Aims: Surgical site infection (SSI) after soft-tissue sarcoma (STS) resection is a serious complication. The purpose of this retrospective study was to investigate the risk factors for SSI after STS resection, and to develop a nomogram that allows patient-specific risk assessment. Methods: A total of 547 patients with STS who underwent tumour resection between 2005 and 2021 were divided into a development cohort and a validation cohort. In the development cohort of 402 patients, the least absolute shrinkage and selection operator (LASSO) regression model was used to screen possible risk factors of SSI. To select risk factors and construct the prediction nomogram, multivariate logistic regression was used. The predictive power of the nomogram was evaluated by receiver operating curve (ROC) analysis in the validation cohort of 145 patients. Results: LASSO regression analysis selected possible risk factors for SSI, including age, diabetes, operating time, skin graft or flap, resected tumour size, smoking, and radiation therapy. Multivariate analysis revealed that age, diabetes, smoking during the previous year, operating time, and radiation therapy were independent risk factors for SSI. A nomogram was developed based on the results of multivariate logistic regression analysis. In the development cohort, the incidence of SSI was 4.5% in the low-risk group (risk score < 6.89) and 26.6% in the high-risk group (risk score ≥ 6.89; p < 0.001). In the validation cohort, the incidence of SSI was 2.0% in the low-risk group and 15.9% in the high-risk group (p = 0.004). Conclusion: Our nomogram will enable surgeons to assess the risk of SSI in patients with STS. In patients with high risk of SSI, frequent monitoring and aggressive interventions should be considered to prevent this.

Targeting Methionine Addiction Combined With Low-dose Irinotecan Arrested Colon Cancer in Contrast to High-dose Irinotecan Alone, Which Was Ineffective, in a Nude-mouse Model.

BACKGROUND/AIM: Colorectal cancer (CRC) is the third-leading cause of death in the world. Although the prognosis has improved due to improvement of chemotherapy, metastatic CRC is still a recalcitrant disease, with a 5-year survival of only 13%. Irinotecan (IRN) is used as first-line chemotherapy for patients with unresectable CRC. However, there are severe side effects, such as neutropenia and diarrhea, which are dose-limiting. We have previously shown that methionine restriction (MR), effected by recombinant methioninase (rMETase), lowered the effective dose of IRN of colon-cancer cells in vitro. The aim of the present study was to evaluate the efficacy of the combination of low-dose IRN and MR on colon-cancer in nude mice. MATERIALS AND METHODS: HCT-116 colon-cancer cells were cultured and subcutaneously injected into the flank of nude mice. After the tumor size reached approximately 100 mm3, 18 mice were randomized into three groups; Group 1: untreated control on a normal diet; Group 2: high-dose IRN on a normal diet (2 mg/kg, i.p.); Group 3: low-dose IRN (1 mg/kg i.p.) on MR effected by a methionine-depleted diet. RESULTS: There was no significant difference between the control mice and the mice treated with high-dose IRN, without MR. However, low-dose IRN combined with MR was significantly more effective than the control and arrested colon-cancer growth (p=0.03). Body weight loss was reversible in the mice treated by low-dose IRN combined with MR. CONCLUSION: The combination of low-dose IRN and MR acted synergistically in arresting HCT-116 colon-cancer grown in nude mice. The present study indicates the MR has the potential to reduce the effective dose of IRN in the clinic.

Tumor-induced Osteomalacia Successfully Treated by En Bloc Tumor Excision and Reconstruction Using a Tumor-bearing Frozen Autograft: A Case Report.

BACKGROUND/AIM: Tumor-induced osteomalacia (TIO) is a rare pathology caused by overproduction of fibroblast growth factor 23 (FGF23). Its common clinical features include generalized muscle weakness, bone pain, and fractures. Complete resection of the offending tumor is the mainstay treatment. In this report, we present the first case of TIO by an FGF23 producing tumor treated using a tumor-bearing autograft treated with liquid nitrogen. CASE REPORT: A 63-year old female presented with generalized body pain, particularly in the left arm. The patient was diagnosed with a FGF23 producing tumor of the left humerus. Wide resection of the involved tumor was performed using a tumor-bearing autograft that was treated with liquid nitrogen. Postoperatively, the FGF23 and alkaline phosphatase (ALP) levels significantly decreased and inorganic phosphate normalized. There was also subsequent relief of generalized body pain. Immediately after the operation, range of motion of the left shoulder and elbow was initiated. The patient was instructed to perform forward flexion and abduction up to 90° with a rotational restraint. Almost complete bone union was observed at 12 months post procedure. Postoperative functional results were as follows: Musculoskeletal Tumor Society (MSTS) score of 27/30, 90% and International Society of Limb Salvage (ISOLS) score of 26/30, 87%. Ten years after the surgery, osteotomy line was completely obscured based on radiographs. The patient was disease free and without activity limitation. CONCLUSION: This is the first case report of wide excision of a FGF23 producing tumor and reconstruction using a tumor-bearing frozen autograft performed with excellent outcomes.

Reduction of Tumor Biomarkers from very High to Normal and Extensive Metastatic Lesions to Undetectability in a Patient With Stage IV HER2-positive Breast Cancer Treated With Low-dose Trastuzumab Deruxtecan in Combination With Oral Recombinant Methioninase and a Low-methionine Diet.

BACKGROUND/AIM: Breast cancer is the most common and the deadliest cancer among women in the world. Treatment options for HER2-positive metastatic breast cancer patients are limited. Trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate (ADC), has recently been introduced as second-line chemotherapy for HER2-positive metastatic breast cancer. The aim of the present study was to evaluate the efficacy of methionine restriction with oral recombinant methioninase (o-rMETase) and a low-methionine diet combined with T-DXd, on a patient with HER2-positive recurrent stage IV breast cancer. CASE REPORT: A 66-year-old female was diagnosed with HER2-positive metastatic breast cancer. Computed tomography (CT) indicated peritoneal dissemination, thickening of the sigmoid colon and splenic flexure and widespread bone metastases. The patient was previously treated with fulvestrant, trastuzumab, pertuzumab, paclitaxel and capecitabine which were ineffective. T-DXd was administered as a second-line chemotherapy. Since the patient experienced strong side effects, the dose of T-Dxd was decreased. The patient began methionine restriction using o-rMETase and a low-methionine diet along with T-DXd. After the start of the combined treatment, CA15-3 and CA27.29, tumor markers for breast cancer, decreased rapidly from a very high level. The levels of both tumor markers are currently normal. Additionally, peritoneal-dissemination nodules, ascites and the thickness of the sigmoid colon and splenic flexure are no longer detected on CT. The patient maintains a high performance status, without severe side effects of the combination treatment. CONCLUSION: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with T-DXd as second-line chemotherapy, was highly effective in a patient with HER2-positive stage IV breast cancer.

Selective Synergy of Rapamycin Combined With Methioninase on Cancer Cells Compared to Normal Cells.

BACKGROUND/AIM: Rapamycin and recombinant methioninase (rMETase) have both shown efficacy to target cancer cells. Rapamycin prevents cancer-cell growth by inhibition of the mTOR protein kinase. rMETase, by degrading methionine, targets the methionine addiction of cancer and has been shown to improve the efficacy of chemotherapy drugs. In the present study, we aimed to determine if a synergy exists between rapamycin and rMETase when used in combination against a colorectal-carcinoma cell line, compared to normal fibroblasts, 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 and Hs-27 human fibroblasts were determined using the CCK-8 Cell Viability Assay. After calculating the IC50 of each drug, we determined the efficacy of rapamycin and rMETase combined on both HCT-116 and Hs-27. RESULTS: Hs-27 normal fibroblasts were more sensitive to rapamycin than HCT-116 colon-cancer cells (IC50=0.37 nM and IC50=1.38 nM, respectively). HCT-116 cells were more sensitive to rMETase than Hs-27 cells (IC50 0.39 U/ml and IC50 0.96 U/ml, respectively). The treatment of Hs-27 cells with the combination of rapamycin (IC50=0.37 nM) and rMETase (IC50=0.96 U/ml) showed no significant difference in their effect on Hs-27 cell viability compared to the two drugs being used separately. However, the treatment of HCT-116 cells with the combination of rapamycin (IC50=1.38 nM) and rMETase (IC50=0.39 U/ml) was able to decrease cancer-cell viability significantly more than either single-drug treatment. CONCLUSION: Rapamycin and rMETase, when used in combination against colorectal-cancer cells, but not normal fibroblasts, in vitro, have a cancer-specific synergistic effect, suggesting that the combination of these drugs can be used as an effective, targeted cancer therapy.

Extensive Synergy Between Recombinant Methioninase and Eribulin Against Fibrosarcoma Cells But Not Normal Fibroblasts.

BACKGROUND/AIM: The aim of the present study was to determine the synergy of recombinant methioninase (rMETase) and the anti-tubulin agent eribulin on fibrosarcoma cells, in comparison to normal fibroblasts, in vitro. MATERIALS AND METHODS: HT1080 human fibrosarcoma cells and HS27 human fibroblasts were used for in vitro experiments. Four groups were analyzed in vitro: No-treatment control; eribulin; rMETase; eribulin plus rMETase. Dual-color HT1080 cells which express red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize cytoplasmic and nuclear dynamics during treatment. RESULTS: Eribulin combined with rMETase greatly decreased the viability of HT 1080 cells. In contrast, eribulin combined with rMETase did not show synergy on Hs27 normal fibroblasts. Eribulin combined with rMETase also caused more fragmentation of the nucleus than all other treatments. CONCLUSION: The combination treatment of eribulin plus rMETase demonstrated efficacy on fibrosarcoma cells in vitro. In contrast, normal fibroblasts were resistant to this combination, indicating the potential clinical applicability of the treatment.

Ten-Year Clinical Outcomes of Endoscope-Assisted Minimally Invasive Surgical Decompression for Lumbar Spinal Stenosis with Degenerative Spondylolisthesis and Comparison with Conservative Treatment.

Introduction: This study aimed to evaluate the 10-year clinical outcomes of endoscope-assisted, minimally invasive surgical (MIS) decompression for lumbar spinal canal stenosis (LSS) with lumbar degenerative spondylolisthesis (DS) and to compare the radiographic changes in patients who underwent this procedure with those who underwent conservative therapy at 10-year follow-up. Methods: Between April 2007 and April 2010, 347 consecutive patients with DS and evidence of LSS underwent conservative treatment first from 2 to 4 weeks. The 114 patients who failed conservative treatment were then treated surgically by endoscope-assisted MIS decompression. Of them, 91 patients were followed for more than 10 years (group S), and 146 of the 233 patients treated conservatively were followed for more than 10 years (group C). Clinical outcomes of endoscope-assisted MIS decompression were assessed using the Short Form Health Survey-36 score (SF-36), the Roland Morris Disability Questionnaire (RDQ), and the neurological leg symptoms of the Japanese Orthopaedic Association Score (JOA score). Radiographic changes of the two groups were assessed by %slip, dynamic %slip, range of motion (ROM), and the height of the disc (DH) on plain radiographs. Results: Significant improvements in clinical outcomes on the SF-36, RDQ, and neurological leg symptoms of the JOA were observed. Radiographic assessment did not show significant differences in the assessed items between the two groups at baseline and after last treatment. Both groups had significantly decreased ROM and DH. Conclusions: The 10-year clinical outcomes of endoscope-assisted MIS decompression for DS were generally good. Furthermore, on radiographic comparison, the progress of spondylolisthesis after this procedure was virtually the same as in the natural course of the disease at 10-year follow-up.

Precise Non-invasive Imaging Mouse Model of Pancreatic Cancer: Very Narrow Band-width Laser Fluorescence Excitation of Green Fluorescent Protein Provides Ultra-bright Tumor Images With no Skin Autofluorescence.

Background/Aim: Pancreatic cancer is a recalcitrant disease with 5-year survival of only 12%. Improved mouse models of pancreatic cancer are critical for discovery of effective therapeutics. Materials and Methods: Orthotopic mouse nude-mouse models of pancreatic cancer were established with the human pancreatic-cancer cell line Panc-1 expressing green fluorescent protein (GFP) by transplanting tumor fragments into the pancreas, using the procedure of surgical orthotopic implantation (SOI). Four weeks after establishment of the orthotopic models, the mice were imaged with the Analytik Jena UVP Biospectrum Advanced with a very-narrow-band-width excitation at 487 nm and peak emission at 513 nm. Results: Non-invasive fluorescence imaging of the mice implanted with Panc-1-GFP showed a very bright tumor in the area of the pancreas and peritoneal cavity. The skin background autofluorescence was absent. When a laparotomy was performed on the mouse for open imaging, the tumor on the pancreas was clearly imaged. There was very clear concordance of the non-invasive image and the image obtained during laparotomy. Conclusion: A precise orthotopic mouse model of pancreatic cancer was developed in which there was high concordance between non-invasive and invasive fluorescence imaging due to the ultra-bright signal and ultra-low background using very-narrow-band-width laser fluorescence excitation. This model can be used for high-throughput in vivo screening for improved therapeutics for pancreatic cancer.

Recombinant Methioninase Decreased the Effective Dose of Irinotecan by 15-fold Against Colon Cancer Cells: A Strategy for Effective Low-toxicity Treatment of Colon Cancer.

BACKGROUND/AIM: Irinotecan (IRN), a topoisomerase I inhibitor and pro-drug of SN-38, is first-line treatment of colon cancer as part of FOLFIRI and FOLFOXIRI combination chemotherapy. However, IRN causes dose-limiting adverse events such as neutropenia and diarrhea. Dose reductions are sometimes required, which reduce efficacy. Recombinant methioninase (rMETase) targets the fundamental basis of cancer, methionine addiction, known as the Hoffman effect, and enhances the efficacy of numerous chemotherapy drugs. The present study determined the efficacy of rMETase when administered in combination with IRN. MATERIALS AND METHODS: Cell viability was assessed by cultivating the HCT-116 human colorectal cancer cell line in 96-well plates at 1×103 cells per well in Dulbecco's modified Eagle's medium (DMEM). Subsequently, HCT-116 cells were treated with increasing concentrations of SN-38, the active form of IRN, ranging from 0.5 nM to 32 nM, and/or rMETase ranging from 0.125 to 8 U/ml. After treatment for 72 h, the half-maximal inhibitory concentration (IC50) of SN-38 alone and rMETase alone for HCT-116 cells were determined. Using the IC50 concentration of rMETase, we determined the IC50 of SN-38 in combination with rMETase. Cell viability was determined with the cell-counting Kit-8 with the WST-8 reagent.. RESULTS: The IC50 of rMETase alone for the HCT-116 cells was 0.55 U/ml, and the IC50 of IRN (SN-38) alone was 3.50 nM. rMETase at 0.55 U/ml lowered the IC50 of SN-38 to 0.232 nM (p<0.0001), a 15-fold reduction. CONCLUSION: rMETase and IRN are strongly synergistic, giving rise to the possibility of lowering the effective dose of IRN for the treatment of patients with colon cancer, thereby reducing its severe toxicity. This new strategy will allow more patients with cancer to be effectively treated with IRN.

[11C] Methionine-PET Imaging as a Cancer Biomarker for Methionine Addiction and Sensitivity to Methionine-restriction-based Combination Chemotherapy.

BACKGROUND/AIM: Methionine addiction is a fundamental and universal hallmark of cancer, termed the Hoffman effect. Methionine addiction of cancer is greater than glucose addiction, termed the Warburg effect, as shown by the comparison of PET imaging with [11C]methionine and [18F]fluorodeoxyglucose. The aim of the present study was to determine whether [11C]methionine PET (MET-PET) images could be a biomarker of methionine addiction of cancer and potential response to methionine-restriction-based combination chemotherapy. PATIENTS AND METHODS: In the present study a patient with invasive lobular carcinoma of the breast metastatic to axillary lymph nodes was imaged by both MET-PET and [18F]fluorodeoxyglucose PET (FDG-PET) before and after combination treatment with methionine restriction, comprising a low-methionine diet and methioninase, along with first-line chemotherapy. RESULTS: MET-PET gave a much stronger and precise image of the patient's metastatic axillary lymph nodes than FDG-PET. The patient had a complete response to methionine restriction-based chemotherapy as shown by MET-PET. CONCLUSION: MET-PET imaging is a biomarker of methionine-addicted cancer and potential response to methionine-restriction-based chemotherapy.

Recombinant Methioninase Lowers the Effective Dose of Regorafenib Against Colon-Cancer Cells: A Strategy for Widespread Clinical Use of a Toxic Drug.

Background/Aim: Regorafenib is a multi-kinase inhibitor, targeting vascular endothelial growth factor receptor 2, fibroblast growth factor receptor 1 and other oncogenic kinases. Regorafenib has efficacy in metastatic colon cancer, but has severe dose-limiting toxicities which cause patients to stop taking the drug. The aim of the present study was to determine if recombinant methioninase (rMETase) could lower the effective concentration of regorafenib in vitro against a colorectal-cancer cell line. Materials and Methods: Firstly, we examined the half-maximal inhibitory concentration (IC50) of regorafenib alone and rMETase alone for the HCT-116 human colorectal-cancer cell line. After that, using the IC50 concentration of each drug, we investigated the efficacy of the combination of regorafenib and rMETase. Results: While both methioninase alone (IC50=0.61 U/ml) and regorafenib alone (IC50=2.26 U/ml) inhibited the viability of HCT-116 cells, the combination of the two agents was more than twice as effective as either alone. Addition of rMETase at 0.61 U/ml lowered the IC50 of regorafenib from 2.26 µM to 1.46 µM. Conclusion: rMETase and regorafenib are synergistic, giving rise to the possibility of lowering the effective dose of regorafenib in patients, thereby reducing its severe toxicity, allowing more cancer patients to be treated with regorafenib.

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.

Case Report: Unresectable pulmonary metastases of a giant cell tumor of bone treated with denosumab: a case report and review of literature.

Giant cell tumors of bone (GCTB) sometimes metastasize to distant organs. In this case report, we present pulmonary metastases of GCTB mimicking malignancies. A 49-year-old man underwent two surgical treatments for a GCTB of the right proximal radius. At the time of the second surgery, no lesions were observed on chest radiography. Three years after surgery, the patient presented with cough and dyspnea, and chest radiography and computed tomography (CT) revealed multiple lung nodules. Positron emission tomography/CT revealed a high accumulation of 18F-fluoro-2-deoxy-D-glucose (18F-FDG) in multiple lesions. Based on the rapid growth and accumulation of 18F-FDG, a metastatic malignant tumor was suspected. CT-guided needle biopsy was performed, and the histology showed proliferation of spindle cells and multinuclear giant cells without malignant changes. Denosumab was administered because multiple lung lesions were unresectable. One month after denosumab treatment, CT showed marked shrinkage of the lesions, and the symptoms significantly improved. Eighteen months after the initial treatment with denosumab, the patient had no symptoms or tumor growth. Although its long-term efficacy and safety remain unclear, denosumab may be a treatment option for patients with unresectable pulmonary GCTB.