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Purpose: The COVID-19 pandemic has influenced various aspects of colorectal cancer (CRC) patient care, including diagnosis, treatment, and outcomes. This study assesses the pandemic's impact on CRC patients. Methods: We performed a retrospective analysis of medical records for CRC patients who underwent surgery at five hospitals affiliated with Hallym University from January 2017 to December 2022. Patients were divided into two groups: the pre-COVID group (2017– 2019) and the COVID group (2020– 2022). Results: Among 2038 patients, 987 (48.4%) were in the pre-COVID group, and 1051 (51.6%) were in the COVID group. The COVID group had more patients with two or more comorbidities (P < 0.001) and a higher incidence of rectal cancer (P = 0.010). While the rates of laparoscopic surgeries were similar, the COVID group had increased emergency surgeries (P = 0.005) and diversion procedures (P = 0.002). Additionally, the COVID group faced more overall complications (P < 0.001) and severe complications (Grade III–V, P = 0.004). There was a rise in lymphovascular invasion (P < 0.001) and T4 stage tumors (P < 0.001) within the COVID group. Despite these differences, both groups had similar 2-year overall survival rates (P = 0.409). Conclusion: Although patients treated during the COVID period experienced more frequent stoma formation, complications, and adverse prognostic factors, there were no differences in short-term oncologic outcomes, which was likely due to the follow-up period being insufficient to detect differences in OS. [ABSTRACT FROM AUTHOR]

Background/Aim: In vivo imaging with luciferaseluciferin has been limited by the inability to visualize the low emitted light, with the signal quantified only by photon counting using a cumbersome highly-cooled CCD camera in a dark room. In the present study, we demonstrate direct visualization of the luciferase-luciferin signal from an orthotopic lung cancer in a nude-mouse xenograft model with a sensitive low-light camera and optics. Materials and Methods: Mouse Lewis-lung carcinoma cells expressing luciferase (LL/2-Luc2) were injected transcutaneously into the lung of a nude mouse. One week later after cell injection, luciferase imaging for emission at 560 nm was performed using the UVP Biospectrum Advanced system after i.v. injection of D-luciferin potassium salt. The intensity of the visualized light was measured and quantified with the instrument. Results: A week following the implantation of LL/2- Luc2 cells in nude mice, the luciferase-luciferin signal from LL/2-Luc2 tumors in the lung was sufficiently visible through the skin to produce true images. At fifteen minutes, the intensity peaked and then progressively dropped due to clearance of luciferin from the tumor. Conclusion: Using the UVP Biospectrum Advanced system we demonstrated non-invasive visualization of true images from luciferase-luciferin signals from an orthotopic lung-cancer mouse model. The luciferase-luciferin emitted light was directly visible through the skin which is a major improvement over previous photon counting to detect the luciferase-luciferin signal. [ABSTRACT FROM AUTHOR]

Background. The objective of this study was to compare the long-term prognosis of patients with T1 and T2 colorectal cancer (CRC) according to lymph node metastasis (LNM) and to identify risk factors for LNM. Methods. We retrospectively reviewed patients who underwent curative resection for T1 or T2 CRC at five University-affiliated hospitals between January 2012 and December 2021. The patients were divided into several groups depending on the presence of LNM or the number of risk factors. Results. Of the total 765 patients, 87 (11.3%) patients had LNM. These patients had poorer recurrence-free survival (RFS) than patients without LNM (72.6% vs. 88.6%). The multivariable analysis showed that high-grade tumors (p = 0.003), lymphovascular invasion (p < 0.001), and rectal location (p = 0.049) were independent predictors of LNM. When divided into groups according to the number of the three risk factors, the risk of LNM increased from 5.4% (ultralow-risk group; no risk factor) to 60.0% (high-risk group; all three risk factors) and the 5-year RFS rate decreased from 96.3% in the ultralow-risk group to 60% in the high-risk group (p < 0.001). Conclusion. Radical surgery should be considered for T1 and T2 CRC patients with these risk factors. [ABSTRACT FROM AUTHOR]

Background/aim: Ivermectin is a widely-used anti-parasitic agent and has shown early promise as an anticancer agent. Recombinant methioninase (rMETase) is a methionine-depleting enzyme targeting the methionine addiction of cancer and has broad efficacy against all tested cancer types. However, the combination efficacy of ivermectin and rMETase on breast cancer cells remains unexplored. The present study aimed to determine the synergistic efficacy of ivermectin and rMETase on MCF-7 human breast cancer cells in vitro., Materials and Methods: The IC 10 of ivermectin and IC 50 of rMETase were determined on MCF-7 cells using the WST-8 reagent to measure cell viability in vitro. MCF-7 cells were treated with four groups: untreated control; ivermectin alone (4.89 μM, IC 10 ); rMETase alone (2.75 U/ml, IC 50 ); and a combination of ivermectin (4.89 μM) and rMETase (2.75 U/ml). Cell viability was assessed 72 hours after treatment with the WST-8 reagent., Results: Treatment with ivermectin (4.89 μM) did not significantly reduce the viability of MCF-7 cells. rMETase (2.75 U/ml) alone significantly reduced MCF-7 cell viability compared to the control group. The combination of ivermectin and rMETase resulted in a significantly greater reduction in cell viability than either agent alone, including a 9.9-fold greater efficacy than ivermectin alone, demonstrating synergistic efficacy (p<0.05)., Conclusion: The combination of ivermectin and rMETase had synergistic efficacy against MCF-7 breast cancer cells in vitro. The present findings suggest that the combination of ivermectin and rMETase is a promising strategy for breast cancer requiring further preclinical and clinical evaluation., (Copyright © 2025 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Chronic lymphocytic leukemia (CLL) is currently incurable. CLL is characterized by disordered DNA methylation. The aim of the present study was to target methylation with methionine restriction in a patient with progressive CLL., Case Report: Methionine restriction for the patient was achieved with a low-methionine vegan diet and oral recombinant methioninase (o-rMETase). The patient also received rituximab, once per week for four weeks, and acalabrutinib 100 mg, twice daily (bid) continuously. The patient's white blood cell count decreased by 95% from peak levels and extensive lymphadenopathy disappeared during combination treatment with o-rMETase, rituximab, and acalabrutinib., Conclusion: The combination of methionine restriction and first-line chemotherapy resulted in an apparent complete response (CR) in a CLL patient, a rare event. The duration of the CR will be monitored, and additional CLL patients will be treated similarly in the future., Competing Interests: The Authors declare no competing interests in relation to this study., (©2025 The Author(s). Published by the International Institute of Anticancer Research.)

Background/aim: Metastatic prostate cancer is a recalcitrant disease. Our laboratory has previously treated prostate-cancer patients with methionine restriction effected by a low methionine diet and oral recombinant methioninase (o-rMETase), both alone and in combination with other agents. The present case is a 66-year-old patient who had a radical prostatectomy in 2019 with a Gleason score 3+3 and 3+4. The patient subsequently was treated with immunotherapy in 2021 and salvage proton-beam therapy in 2022, and subsequently treated only with o-rMETase and a low-methionine diet. The aim of the present study was to determine the long-term efficacy of methionine restriction on the patient's prostate cancer., Case Report: Starting in September 2022, the patient started methionine restriction with a low methionine-diet and o-rMETase, twice a day, after meals, at 250 units/dose. Since the start of methionine restriction, the patients' prostate-specific antigen (PSA) has remained stable, under 2 ng/ml. Positron emission tomography/computed tomography (PET/CT) and prostate specific membrane antigen (PSMA)-PET imaging indicated in September 2023 a right pelvic-side-wall metastatic lymph node that was stable when the PSMA-PET scan was repeated in March 2024, with the standardized uptake value (SUV) decreasing from 19.39 to 14.98. A very small possible metastatic external-iliac lymph node was detected in March 2024. Thus, the lymph-node metastases were stable and did not increase., Conclusion: During the time the patient was on methionine restriction alone, effected by a low-methionine diet and o-rMETase, the metastatic prostate cancer did not progress. Further clinical studies of methionine restriction and metastatic prostate cancer are needed, including randomized clinical trials., Competing Interests: The Authors declare no competing interests., (©2025 The Author(s). Published by the International Institute of Anticancer Research.)

Background/aim: For second-line chemotherapy of soft-tissue sarcoma, gemcitabine is administered in combination with docetaxel. However, more effective treatments are required for advanced soft-tissue sarcoma, where the efficacy is limited. The purpose of the present study was to compare the efficacy of rMETase and gemcitabine against HT1080 human fibrosarcoma cells and Hs27 normal fibroblasts, as well as to identify and effectively treat HT1080 cells that are resistant to gemcitabine associated with elevated c-MYC., Patients and Methods: Cell viability was measured with the WST-8 reagent. Four groups of in vitro tests were conducted involving HT1080 and Hs27 cells: gemcitabine alone, rMETase alone, and a combination of gemcitabine plus rMETase. Gemcitabine resistant cells (GR-HT1080) were established by culturing HT-1080 cells in increasing concentrations of gemcitabine, ranging from 0.016 nM to 16 nM over five months. Western immunoblotting was performed to measure c-MYC levels in HT1080 and GR-HT1080 cells., Results: Gemcitabine had an IC 50 of 12.8 nM against HT1080 cells, 30.8 nM against GR-HT1080 cells, and 4.48 nM against Hs27 cells. The rMETase IC 50 value for HT1080 was 0.75 U/ml. The IC 50 value of rMETase for GR-HT1080 cells was 0.85 U/ml. The IC 50 value for rMETase on Hs27 cells was 0.93 U/ml. Gemcitabine and rMETase demonstrated synergy in killing fibrosarcoma cells, but no synergy was observed on normal fibroblasts. The c-MYC level that was more than 5.1 times higher in GR-HT1080 cells compared to HT-1080 cells. Both the parental HT1080 cells and the GR-HT1080 cells had a similar high sensitivity to rMETase alone., Conclusion: rMETase may be used as a future clinical strategy to overcome gemcitabine resistance in sarcoma., Competing Interests: The Authors declare no competing interests in relation to this study., (©2025 The Author(s). Published by the International Institute of Anticancer Research.)

Background/aim: Salmonella typhimurium A1-R (A1-R) targets and inhibits a wide range of cancer types without continuously infecting healthy tissue. Chloroquine, an antimalarial drug, induces apoptosis and inhibits autophagy in cancer cells. The aim of the present study was to determine the synergy of A1-R plus chloroquine on HT1080 human fibrosarcoma cells in vitro and in a nude-mouse model., Materials and Methods: HT1080 human fibrosarcoma cells were used for in vitro experiments. Four groups were analysed in vitro: No-treatment control; A1-R; chloroquine; A1-R plus chloroquine. The nude-mouse models of HT1080 human fibrosarcoma were randomly assigned into four groups: G1: untreated control; G2: Oral A1-R [5×10 7 colony forming units (CFU)/body, twice a week, 2 weeks]; G3: Chloroquine [100 mg/kg/body, intraperitoneal (IP) administration, twice a week, 2 weeks]; G4: Oral A1-R (5×10 7 CFU/body), twice a week, 2 weeks plus chloroquine (100 mg/kg/body, IP), twice a week, 2 weeks. Each cohort consisted of five mice. Tumor volume and body weight were assessed biweekly., Results: A1-R combined with chloroquine synergistically decreased the viability of HT1080 cells in vitro compared to other groups. Orally-administered A1-R at 5×10 7 CFU combined with IP-administered chloroquine eradicated HT1080 tumors in nude mice, without body-weight decrease., Conclusion: The combination treatment of A1-R plus chloroquine demonstrated synergy against HT1080 cancer cells in vitro and in vivo. A1-R was administered orally, suggesting its potential as a probiotic. The present results suggest the clinical potential of the combination of A1-R and chloroquine for soft-tissue sarcoma therapy, a recalcitrant disease., (Copyright © 2025, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Ivermectin was initially utilized as a veterinary medication, demonstrating efficacy against various parasites. Pancreatic cancer is currently one of the most recalcitrant diseases. The aim of the present study was to demonstrate the synergy of the combination of recombinant methioninase (rMETase) and ivermectin to eradicate human pancreatic cancer cells in vitro., Materials and Methods: MiaPaCa-2 human pancreatic cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with the addition of 10% fetal bovine serum and 1 IU/ml penicillin/streptomycin. Reduction of cell viability by rMETase alone and ivermectin alone and their combination on MiaPaCa-2 cells was determined with the WST-reagent. Four experimental groups were examined in vitro: control group without treatment; ivermectin alone; rMETase alone; ivermectin combined with rMETase., Results: The IC 50 of ivermectin for MiaPaCa-2 cells was 5.9 μM. The IC 50 of rMETase on MiaPaCa-2 cells was 2.93 U/ml. Ivermectin (5.9 μM) plus rMETase (2.93 U/ml) synergistically greatly reduced the viability of MiaPaCa-2 cells, compared to ivermectin alone (80% reduction vs. 45% reduction, respectively p<0.05)., Conclusion: The combination of ivermectin and rMETase effectively eradicated MiaPaCa-2 pancreatic cancer cells. The present results indicate the future clinical potential of the combination of rMETase, currently administered orally to patients as a dietary supplement, and oral ivermectin on pancreatic cancer., (Copyright © 2025 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Methionine addiction, known as the Hoffman effect, makes cancer cells more sensitive to methionine restriction than normal cells. However, the long-term effects of methionine restriction on cancer and normal cells have not been thoroughly studied., Materials and Methods: HCT-116 human colorectal-cancer cells and Hs27 normal skin fibroblasts were treated with 0-8 U/ml of recombinant methioninase (rMETase) for 12 days. The cells were cultured in Dulbecco's modified Eagle's medium in 96-well tissue-culture plates., Results: HCT-116 cells were sensitive to all concentrations of rMETase from 0.125 U/ml to 8 U/ml. After day-8 of treatment, HCT-116 cells were acutely sensitive to rMETase, especially at rMETase concentrations of 0.5 U/ml or higher. Normal Hs27 fibroblasts were much less sensitive to rMETase: In the range of 0.125 U/ml to 0.5 U/ml, rMETase had no effect on Hs27 cells. rMETase concentrations up to 2 U/ml had a slight initial effect on Hs27 cells, whereas at concentrations ranging from 4 U/ml to 8 U/ml, rMETase reduced Hs27 viability over the 12-day test period, with acute loss of viability observed after eight days of exposure., Conclusion: Cancer cells were significantly more sensitive to rMETase than normal cells, with an acute loss of cell viability observed in cancer cells after eight days of treatment at concentrations of 0.5 U/ml or higher. These findings highlight the large difference in sensitivity between cancer and normal cells to rMETase and introduce the phenomenon of acute cell death in methionine restriction, which we term "methionine-depletion catastrophe"., (Copyright © 2025 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Ifosfamide is used clinically with doxorubicin as first-line chemotherapy for soft-tissue sarcoma. However, ifosfamide efficacy for soft-tissue sarcoma is limited due to frequent occurence of ifosfamide resistance and thus more effective therapy is needed. The present study aimed to determine the synergy of recombinant methioninase (rMETase) plus ifosfamide against HT1080 human fibrosarcoma cells in vitro. Additionally, the present study also investigated the efficacy of a methionine-restricted diet combined with ifosfamide in nude-mouse models of ifosfamide-resistant HT1080 (IR-HT1080)., Materials and Methods: Cell viability for HT1080 human fibrosarcoma cells was determined in four groups in vitro: No treatment control; ifosfamide alone; rMETase alone; and a combination of ifosfamide plus rMETase. HT1080 tumors were established in nude mice subcutaneously. The HT1080 tumor models were treated by administering ifosfamide by intraperitoneal injection twice a week, for a total of 11 doses. Surviving tumors were considered ifosfamide resistant (IR-HT1080). Four groups of IR-HT1080 nude-mouse models were subsequently established: Group 1 was a no-treatment control, Group 2 received ifosfamide, Group 3 was given a methionine-restricted diet (MR), and Group 4 received ifosfamide plus MR. Additionally, two groups of nude mice with parental HT1080 subcutaneous tumors were included: Group 5 was a no-treatment control, and Group 6 received ifosfamide for comparison., Results: The 50% inhibitory concentration (IC 50 ) for ifosfamide against HT1080 cells was 0.38 mM. The IC 50 for rMETase was 0.75 U/ml for HT1080 cells (data from [4]). The combination of rMETase (0.75 U/ml) plus ifosfamide (0.38 mM) was synergistic against HT1080 fibrosarcoma cells in vitro. The combination of ifosfamide plus MR eradicated the IR-HT1080 tumors in nude-mouse models, while each treatment alone achieved limited tumor inhibition., Conclusion: The present results suggest the combination of MR and ifosfamide has promising potential for overcoming ifosfamide resistance in future clinical applications., (Copyright © 2025, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The surgical environment can influence the clinical outcomes of procedures and patient conditions. This retrospective study aimed to evaluate how surgical timing affects short-term outcomes in emergency laparoscopic appendectomy for acute appendicitis. Methods: A total of 647 patients with acute appendicitis who underwent emergency laparoscopic appendectomy at Chuncheon Sacred Heart Hospital between January 2018 and June 2021 were included in this study. The study cohort was divided into the following two groups based on the timing of surgery: work hours and out-of-hours (weekends, holidays, or weekday nights). Clinical outcomes were then compared between the groups. Results: Work-hour and out-of-hours appendectomies were performed in 282 and 365 patients, respectively. Baseline characteristics and types of appendicitis were similar between the groups (complicated appendicitis: 26.6% in the work-hours group versus 30.4% in the out-of-hours group, P = .288). Operation times were comparable (35.10 minutes versus 34.33 minutes, P = .620), with no cases requiring conversion to open appendectomy in either group. The overall rate of 30-day postoperative complications did not differ significantly between the groups (7.8% versus 10.4%, P = .849). The severity of postoperative complications, categorized by the modified Clavien-Dindo classification, did not show significant differences between the groups ( P = .849). In addition, the time to functional recovery was similar in both groups. Conclusions: The clinical outcomes of out-of-hours laparoscopic appendectomy were similar to those of procedures performed during working hours. Therefore, scheduling emergency surgery can be determined based on the patient's condition and the hospital's capacity to manage acute appendicitis.

Background/aim: Docetaxel combined with gemcitabine is a second-line treatment for soft-tissue sarcoma; however, its effectiveness is limited because of docetaxel resistance. The objective of the present study was to determine the potential of recombinant methioninase (rMETase) to enhance the efficacy of docetaxel on high-docetaxel-resistant human fibrosarcoma cells in vitro., Materials and Methods: Docetaxel-resistant HT1080 (DTR-HT1080) human fibrosarcoma cells were established by culturing them in by progressively increasing concentrations of docetaxel from 0.02 to 9 nM in vitro. The IC 50 values for docetaxel and rMETase, as well as the efficacy of their combination, in inhibiting HT1080 human fibrosarcoma cells, DTR-HT1080 cells, and Hs27 normal human fibroblasts were determined. Four experimental groups were examined in vitro: control group without treatment; docetaxel alone; rMETase alone; docetaxel combined with rMETase., Results: The IC 50 of docetaxel for DTR-HT1080 cells was 7.57 nM, compared to the parental HT1080 cells with an IC 50 of 1.68 nM, a 4.5-fold increase. The IC 50 of docetaxel on Hs27 fibroblasts was 4.46 nM. The IC 50 of rMETase on HT1080 cells was 0.75 U/ml (data from [6]). The IC 50 of rMETase on DTR-HT1080 cells was 0.55 U/ml. The IC 50 of rMETase on Hs27 fibroblasts was 0.93 U/ml (data from [6]). Docetaxel (1.68 nM [IC 50 ]) plus rMETase (0.75 U/ml [IC 50 ]) synergistically reduced the viability of HT1080 cells (p<0.05). In contrast, docetaxel (4.46 nM) plus rMETase (0.93 U/ml) did not reduce the viability of Hs27 fibroblasts, compared to either agent alone. The combination of rMETase (0.55 U/ml [IC 50 ]) and docetaxel (1.68 nM [IC 50 of the parental cells]) overcame docetaxel resistance of DTR-HT1080 cells, resulting in an inhibition of 48.1% compared to docetaxel alone (6.8%) or rMETase alone (37.5%) (p<0.05). rMETase thus increased the efficacy of docetaxel 7-fold on docetaxel-resistant human fibrosarcoma cells., Conclusion: The combination of docetaxel and rMETase was synergistic on HT1080 fibrosarcoma cells, but not normal fibroblasts. rMETase plus docetaxel synergistically reduced the high docetaxel resistance of DTR-HT1080 cells. The present results indicate the clinical potential of rMETase to reduce docetaxel resistance in soft-tissue sarcoma patients in the future., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Salmonella typhimurium A1-R has been shown to target and inhibit many types of cancers in mouse models without continuous infection of normal tissue. The objective of the present study was to determine the effective dose of orally-administered Salmonella typhimurium A1-R, expressing-green fluorescent protein (GFP), on an HT1080 human-fibrosarcoma nude-mouse model., Materials and Methods: The HT1080-human- fibrosarcoma nude-mouse models were randomized into the following three groups: G1: untreated control; G2: Oral Salmonella typhimurium A1-R (5×10 7 colony forming units [CFU]/body, twice a week, 2 weeks); G3: Oral Salmonella typhimurium A1-R (3.3×10 8 CFU/body, twice a week, 2 weeks). Each group comprised five mice. Body weight and tumor volume were measured twice a week. The number of colonies of Salmonella typhimurium A1-R-GFP in excised tumors and excised livers in groups G2 and G3 were determined on day 3, day 7 and 14 by growth on agar plates. Tukey-Kramer analysis was used to examine the relationships between variables. Statistically-significant results are defined as those with p≤0.05., Results: Salmonella typhimurium A1-R was administered orally at a dose of 3.3×10 8 CFU, which successfully regressed the HT1080 tumor in nude mice. However, this effect was not observed at a lower dose of 5×10 7 CFU. After administering Salmonella typhimurium A1-R at 3.3×10 8 CFU, tumors and liver tissues were harvested, homogenized, and cultured on days 3, 7 and 14. Resulting GFP-expressing Salmonella typhimurium A1-R colonies were then counted. The number of GFP-bacterial colonies derived from excised tumors at intervals of 3, 7, and 14 days increased over time post-administration of oral GFP-Salmonella typhimurium. Conversely, the number of GFP-Salmonella typhimurium A1-R colonies that could be grown from excised livers decreased over time, following oral administration of GFP-Salmonella typhimurium. Additionally, the GFP-bacterial colonies grown from the excised tumors were significantly larger than those grown from the excised livers., Conclusion: The present study showed that an aggressive fibrosarcoma could be regressed by orally-administered Salmonella typhimurium A1-R which accurately targeted tumors without continuous growth in normal organs. The present results suggested the potential of orally-administered Salmonella typhimurium A1-R as a probiotic to treat aggressive soft-tissue sarcoma., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Methionine restriction selectively arrests cancer cells during the S-phase of the cell cycle. We hypothesized that DNA damage may occur in S-phase in cancer cells during methionine restriction. To determine if this occurs, we used MiaPaCa-2 Tet-On 53BP1-green fluorescent protein (GFP) pancreatic cancer cells, which report GFP fluorescence in real time after DNA-damage response (DDR) in these cells. We also determined whether a chemotherapy drug in combination with methionine restriction increases the rate of DNA damage., Materials and Methods: MiaPaCa-2 Tet-On 53BP1-GFP cells were used for in vitro experiments. The 25% and 50% inhibitory concentrations (IC 25 and IC 50 , respectively) of recombinant methioninase (rMETase) and paclitaxel on MiaPaCa-2 Tet-On 53BP1-GFP pancreatic cancer cells were determined. Cell viability and DDR with rMETase alone, paclitaxel alone, and their combination were measured in MiaPaCa-2 Tet-On 53BP1-GFP cells., Results: The IC 25 of rMETase on MiaPaCa-2 Tet-On 53BP1-GFP cells was 1.66 U/ml. The IC 25 for paclitaxel on MiaPaCa-2 Tet-On 53BP1-GFP cells was 3.31 nM. The combination of rMETase and paclitaxel synergistically reduced the viability of MiaPaCa-2 Tet-On 53BP1-GFP cells. The IC 50 of paclitacel on MiaPaCa-2 Tet-On 53BP1-GFP cells was 5.1 nM. The IC 50 of rMETase on MiaPaCa-2 Tet-On 53BP1-GFP cells was 2.3 U/ml. The combination of rMETase (IC 50 ) plus paclitaxel (IC 50 ) on MiaPaCa-2 Tet-On 53BP1-GFP cells also caused more DNA damage than either agent alone., Conclusion: The present study suggests the synergy of methionine restriction and chemotherapy is due, at least in part, to DNA damage of cancer cells., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) were previously developed by our laboratory, AntiCancer Inc. In the present study, we demonstrate imaging of the GFP, RFP, or CFP nude mice with single-nanometer-tuning laser fluorescence excitation with a single instrument., Materials and Methods: Female transgenic C57/B6 nude GFP, RFP, and CFP mice aged six weeks were used. The images were obtained using the UVP Biospectrum Advanced system (Analytik Jena US LLC) with excitation at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP., Results: For each color transgenic fluorescent mouse, images without background could be obtained individually with the UVP Biospectrum Advanced system., Conclusion: Using a single instrument, brilliant and well-defined images of GFP, RFP, and CFP mice were obtained with single-nanometer-tuning laser fluorescence excitation. This imaging system will be used in future studies to analyze cancer cells in the colored mice that are spectrally distinct in order to determine how stromal cells and cancer interact in the tumor microenvironment., Competing Interests: AW, NC, and SG are employees of Analytik Jena. JR is an employee of Anticancer Inc. KM, SM, MS, BMK, and RMH are non-salaried associates of AntiCancer Inc. AntiCancer Inc. uses mouse models of cancer for contract research., (©2024 The Author(s). Published by the International Institute of Anticancer Research.)

Background/aim: A major challenge in treating soft-tissue sarcoma is the development of drug resistance. Eribulin, an anti-tubulin agent, is used as a second-line chemotherapy for patients with unresectable or metastatic soft-tissue sarcoma. However, most patients with advanced soft-tissue sarcoma are resistant to eribulin and do not survive. Recombinant methioninase (rMETase) targets the fundamental and general hallmark of cancer, methionine addiction, termed the Hoffman Effect. The present study aimed to show how much rMETase could increase the efficacy of eribulin on eribulin-resistant fibrosarcoma cells in vitro., Materials and Methods: HT1080 human fibrosarcoma cells were exposed to step-wise increasing concentrations of eribulin from 0.15-0.4 nM to establish eribulin-resistant HT1080 (ER-HT1080). ER-HT1080 cells were cultured in vitro and divided into four groups: untreated control; eribulin treated (0.15 nM); rMETase treated (0.75 U/ml); and eribulin (0.15 nM) plus rMETase (0.75 U/ml) treated., Results: The IC 50 of eribulin on ER-HT1080 cells was 0.95 nM compared to the IC 50 of 0.15 nM on HT1080 cells, a 6-fold increase. The IC 50 of rMETase on ER-HT1080 and HT1080 was 0.87 U/ml and 0.75 U/ml, respectively. The combination of rMETase (0.75 U/ml) and eribulin (0.15 nM) was synergistic on ER-HT1080 cells resulting in an inhibition of 80.1% compared to eribulin alone (5.0%) or rMETase alone (47.1%) (p<0.05). rMETase thus increased the efficacy of eribulin 16-fold on eribulin-resistant fibrosarcoma cells., Conclusion: The present study showed that the combination of eribulin and rMETase can overcome high eribulin resistance of fibrosarcoma. The present results demonstrate that combining rMETase with first- or second-line therapy for soft-tissue sarcoma has the potential to overcome the intractable clinical problem of drug-resistant soft-tissue sarcoma., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Pancreatic cancer has a very poor prognosis with a 5-year survival rate of less than 5% among patients with distant metastasis, a figure that has not improved over many decades. Only 10 to 20% patients are candidates for curative surgery at presentation due to the aggressive nature and asymptomatic progression of pancreatic cancer. Although first-line chemotherapy, such as FOLFIRINOX and gemcitabine + nab paclitaxel, improved the median survival from 8.5 to 11.1 months, more effective treatments are immediately needed. The aim of the present study was to evaluate the efficacy of methionine restriction with oral rMETase (o-rMETase) and a low-methionine diet combined with first-line chemotherapy on a patient with stage IV metastatic pancreatic cancer., Case Report: A 63-year-old female was diagnosed with metastatic pancreatic cancer in October 2023. The patient started FOLFIRINOX as first-line chemotherapy in combination with methionine restriction, which comprised o-rMETase 250 units twice a day and a low-methionine diet. The patient was monitored using computed tomography and CA19-9 blood tests. After five months from the start of combination therapy, the size of the primary tumor decreased by 40% along with liver-metastasis regression. The CA19-9 blood marker decreased by 86%. The patient sustains a high performance status and continues the combination therapy without severe side effects., Conclusion: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with first-line chemotherapy, was highly effective in a patient with inoperable stage IV pancreatic cancer., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Positron emission tomography (PET) is an important imaging modality, especially in oncology. [ 18 F]fluorodeoxyglucose PET (FDG-PET) is the most used cancer PET imaging. However, since the elevated glucose use by cancers, termed the Warburg effect, is usually only moderate, FDG often does not provide a strong or well-delineated signal. Malignancies have a stronger addiction to methionine, known as the Hoffman effect, and thus [ 11 C]methionine PET (MET-PET) has demonstrated superiority over FDG-PET in gliomas and other brain tumors. Our team is pioneering the use of MET-PET for tumors of the trunk for both better detection of cancer and to determine candidates for methionine-restriction therapy. The present study provides examples of cancers of organs in the trunk in which MET-PET outperforms FDG-PET in detecting and delineating primary and metastatic cancer., Patients and Methods: In all cases, MET-PET and FDG-PET were performed simultaneously. An evaluation of the images was conducted by a nuclear medicine physician., Results: Four cases, including prostate, bladder, esophageal, and breast cancer demonstrated the superiority of MET-PET compared to FDG-PET., Conclusion: MET-PET can out-perform FDG PET for accurate detection of primary and metastatic cancer in the trunk and can determine the extent of methionine addiction of cancer, thereby indicating whether cancer patients can benefit from methionine-restriction therapy., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Doxorubicin is first-line therapy for soft-tissue sarcoma, but patients can develop resistance which is usually fatal. As a novel therapeutic strategy, the present study aimed to determine the synergy of recombinant methioninase (rMETase) and doxorubicin against HT1080 fibrosarcoma cells compared to Hs27 normal fibroblasts, and rMETase efficacy against doxorubicin-resistant HT1080 cells in vitro., Materials and Methods: The 50% inhibitory concentrations (IC 50 ) of doxorubicin and rMETase, as well as their combination efficacy, against HT1080 human fibrosarcoma cells, Hs27 normal human fibroblasts and doxorubicin-resistant HT1080 (DR-HT1080) cells were determined. Dual-color HT1080 cells which expressed red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize nuclear fragmentation during treatment. Nuclear fragmentation was observed with an IX71 fluorescence microscope., Results: The IC 50 for doxorubicin was 3.3 μM for HT1080 cells, 12.4 μM for DR-HT1080 cells, and 7.25 μM for Hs27 cells. The IC 50 for rMETase was 0.75 U/ml for HT1080 cells, 0.42 U/ml for DR-HT1080 cells, and 0.93 U/ml for Hs27 cells. The combination of rMETase and doxorubicin was synergistic against fibrosarcoma cells but not against normal fibroblasts. The combination of doxorubicin plus rMETase also caused more fragmented nuclei than either treatment alone in HT1080 cells. rMETase alone was highly effective against the DR-HT1080 cells as well as the parental HT1080 cells., Conclusion: The present results indicate the future clinical potential of rMETase in combination with doxorubicin for fibrosarcoma, including doxorubicin-resistant fibrosarcoma., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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 (IC 50 ) 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 IC 50 s for PC-3 was determined., Results: The IC 50 of docetaxel for PC-3 and for Hs27 was 0.72 nM and 0.94 nM, respectively. The IC 50 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., Competing Interests: There are no conflicts of interest, according to the Authors., (Copyright 2024, International Institute of Anticancer Research.)

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 (IC 50 ) value of rMETase on HCT-116 human colorectal cancer cells. HCT-116 cells were treated with rMETase at the IC 50 Western immunoblotting was used to compare PD-L1 expression in HCT-116 cells treated with and without rMETase., Results: The IC 50 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., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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/G 2 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., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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 1×10 3 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 (IC 50 ) of SN-38 alone and rMETase alone for HCT-116 cells were determined. Using the IC 50 concentration of rMETase, we determined the IC 50 of SN-38 in combination with rMETase. Cell viability was determined with the cell-counting Kit-8 with the WST-8 reagent.., Results: The IC 50 of rMETase alone for the HCT-116 cells was 0.55 U/ml, and the IC 50 of IRN (SN-38) alone was 3.50 nM. rMETase at 0.55 U/ml lowered the IC 50 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., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)