Selective methioninase-induced trap of cancer cells in S/G2 phase visualized by FUCCI imaging confers chemosensitivity.

A major impediment to the response of tumors to chemotherapy is that the large majority of cancer cells within a tumor are quiescent in G0/G1, where cancer cells are resistant to chemotherapy. To attempt to solve this problem of quiescent cells in a tumor, cancer cells were treated with recombinant methioninase (rMETase), which selectively traps cancer cells in S/G2. The cell cycle phase of the cancer cells was visualized with the fluorescence ubiquitination-based cell cycle indicator cell cycle indicator (FUCCI). At the time of rMETase-induced S/G2-phase blockage, identified by the cancer cells' green fluorescence by FUCCI imaging, the cancer cells were administered S/G2-dependent chemotherapy drugs, which interact with DNA or block DNA synthesis such as doxorubicin, cisplatin, or 5-fluorouracil. Treatment of cancer cells with drugs only, without rMETase-induced S/G2 phase blockage, led to the majority of the cancer-cell population being blocked in G0/G1 phase, identified by the cancer cells becoming red fluorescent in the FUCCI system. The G0/G1 blocked cells were resistant to the chemotherapy. In contrast, trapping of cancer cells in S/G2 phase by rMETase treatment followed by FUCCI-imaging-guided chemotherapy was highly effective in killing the cancer cells

Toxicology and efficacy of tumor-targeting Salmonella typhimurium A1-R compared to VNP 20009 in a syngeneic mouse tumor model in immunocompetent mice.

Salmonella typhimurium A1-R (S. typhimurium A1-R) attenuated by leu and arg auxotrophy has been shown to target multiple types of cancer in mouse models. In the present study, toxicologic and biodistribution studies of tumor-targeting S. typhimurium A1-R and S. typhimurium VNP20009 (VNP 20009) were performed in a syngeneic tumor model growing in immunocompetent BALB/c mice. Single or multiple doses of S. typhimurium A1-R of 2.5 × 105 and 5 × 105 were tolerated. A single dose of 1 × 106 resulted in mouse death. S. typhimurium A1-R (5 × 105 CFU) was eliminated from the circulation, liver and spleen approximately 3-5 days after bacterial administration via the tail vein, but remained in the tumor in high amounts. S. typhimurium A1-R was cleared from other organs much more rapidly. S. typhimurium A1-R and VNP 20009 toxicity to the spleen and liver was minimal. S. typhimurium A1-R showed higher selective targeting to the necrotic areas of the tumors than VNP20009. S. typhimurium A1-R inhibited the growth of CT26 colon carcinoma to a greater extent at the same dose of VNP20009. In conclusion, we have determined a safe dose and schedule of S. typhimurium A1-R administration in BALB/c mice, which is also efficacious against tumor growth. The results of the present report indicate similar toxicity of S. typhimurium A1-R and VNP20009, but greater antitumor efficacy of S. typhimurium A1-R in an immunocompetent animal. Since VNP2009 has already proven safe in a Phase I clinical trial, the present results indicate the high clinical potential of S. typhimurium A1-R

Combination treatment with recombinant methioninase enables temozolomide to arrest a BRAF V600E melanoma in a patient-derived orthotopic xenograft (PDOX) mouse model.

An excessive requirement for methionine termed methionine dependence, appears to be a general metabolic defect in cancer. We have previously shown that cancer-cell growth can be selectively arrested by methionine deprivation such as with recombinant methioninase (rMETase). The present study used a previously-established patient-derived orthotopic xenograft (PDOX) nude mouse model of BRAF V600E-mutant melanoma to determine the efficacy of rMETase in combination with a first-line melanoma drug, temozolomide (TEM). In the present study 40 melanoma PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n=10); TEM (25 mg/kg, oral 14 consecutive days, n=10); rMETase (100 units, intraperitoneal 14 consecutive days, n=10); combination TEM + rMETase (TEM: 25 mg/kg, oral rMETase: 100 units, intraperitoneal 14 consecutive days, n=10). All treatments inhibited tumor growth compared to untreated control (TEM: p=0.0081, rMETase: p=0.0037, TEM-rMETase: p=0.0024) on day 14 after initiation. However, the combination therapy of TEM and rMETase was significantly more efficacious than either mono-therapy (TEM: p=0.0051, rMETase: p=0.0051). The present study is the first demonstrating the efficacy of rMETase combination therapy in a PDOX model, suggesting potential clinical development, especially in recalcitrant cancers such as melanoma, where rMETase may enhance first-line therapy

Recombinant methioninase (rMETase) is an effective therapeutic for BRAF-V600E-negative as well as -positive melanoma in patient-derived orthotopic xenograft (PDOX) mouse models.

Melanoma is a recalcitrant disease. Melanoma patients with the BRAF-V600E mutation have been treated with the drug vemurafenib (VEM) which targets this mutation. However, we previously showed that VEM is not very effective against a BRAF-V600E melanoma mutant in a patient-derived orthotopic xenograft (PDOX) model. In contrast, we demonstrated that recombinant methioninase (rMETase) which targets the general metabolic defect in cancer of methionine dependence, was effective against the BRAF-V600E mutant melanoma PDOX model. In the present study, we demonstrate that rMETase is effective against a BRAF-V600E-negative melanoma PDOX which we established. Forty BRAF-V600E-negative melanoma PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); temozolomide (TEM) (25 mg/kg, p.o., 14 consecutive days, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); TEM + rMETase (TEM: 25 mg/kg, p.o., rMETase: 100 units, i.p., 14 consecutive days, n = 10). All treatments inhibited tumor growth compared to untreated control (TEM: p = 0.0003, rMETase: p = 0.0006, TEM/rMETase: p = 0.0002) on day 14 after initiation. Combination therapy of TEM and rMETase was significantly more effective than either mono-therapy (TEM: p = 0.0113, rMETase: p = 0.0173). The present study shows that TEM combined with rMETase is effective for BRAF-V600E-negative melanoma PDOX similar to the BRAF-V600E-positive mutation melanoma. These results suggest rMETase in combination with first-line chemotherapy can be highly effective in both BRAF-V600E-negative as well as BRAF-V600E-positive melanoma and has clinical potential for this recalcitrant disease

Intra-tumor L-methionine level highly correlates with tumor size in both pancreatic cancer and melanoma patient-derived orthotopic xenograft (PDOX) nude-mouse models.

An excessive requirement for methionine (MET) for growth, termed MET dependence, appears to be a general metabolic defect in cancer. We have previously shown that cancer-cell growth can be selectively arrested by MET restriction such as with recombinant methioninase (rMETase). In the present study, we utilized patient-derived orthotopic xenograft (PDOX) nude mouse models with pancreatic cancer or melanoma to determine the relationship between intra-tumor MET level and tumor size. After the tumors grew to 100 mm3, the PDOX nude mice were divided into two groups: untreated control and treated with rMETase (100 units, i.p., 14 consecutive days). On day 14 from initiation of treatment, intra-tumor MET levels were measured and found to highly correlate with tumor volume, both in the pancreatic cancer PDOX (p<0.0001, R2=0.89016) and melanoma PDOX (p<0.0001, R2=0.88114). Tumors with low concentration of MET were smaller. The present results demonstrates that patient tumors are highly dependent on MET for growth and that rMETase effectively lowers tumor MET

Recombinant methioninase combined with doxorubicin (DOX) regresses a DOX-resistant synovial sarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model.

Synovial sarcoma (SS) is a recalcitrant subgroup of soft tissue sarcoma (STS). A tumor from a patient with high grade SS from a lower extremity was grown orthotopically in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) mouse model. The PDOX mice were randomized into the following groups when tumor volume reached approximately 100 mm3: G1, control without treatment; G2, doxorubicin (DOX) (3 mg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks; G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G4 DOX (3mg/kg), i.p. weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks). On day 14 after treatment initiation, all therapies significantly inhibited tumor growth compared to untreated control, except DOX: (DOX: p = 0.48; rMETase: p < 0.005; DOX combined with rMETase < 0.0001). DOX combined with rMETase was significantly more effective than both DOX alone (p < 0.001) and rMETase alone (p < 0.05). The relative body weight on day 14 compared with day 0 did not significantly differ between any treatment group or untreated control. The results indicate that r-METase can overcome DOX-resistance in this recalcitrant disease

Recombinant methioninase effectively targets a Ewing's sarcoma in a patient-derived orthotopic xenograft (PDOX) nude-mouse model.

Methionine dependence is due to the overuse of methionine for aberrant transmethylation reactions in cancer. Methionine dependence may be the only general metabolic defect in cancer. In order to exploit methionine dependence for therapy, our laboratory previously cloned L-methionine α-deamino-γ-mercaptomethane lyase [EC 4.4.1.11]). The cloned methioninase, termed recombinant methioninase, or rMETase, has been tested in mouse models of human cancer cell lines. Ewing's sarcoma is recalcitrant disease even though development of multimodal therapy has improved patients'outcome. Here we report efficacy of rMETase against Ewing's sarcoma in a patient-derived orthotopic xenograft (PDOX) model. The Ewing's sarcoma was implanted in the right chest wall of nude mice to establish a PDOX model. Eight Ewing's sarcoma PDOX mice were randomized into untreated control group (n = 4) and rMETase treatment group (n = 4). rMETase (100 units) was injected intraperitoneally (i.p.) every 24 hours for 14 consecutive days. All mice were sacrificed on day-15, 24 hours after the last rMETase administration. rMETase effectively reduced tumor growth compared to untreated control. The methionine level both of plasma and supernatants derived from sonicated tumors was lower in the rMETase group. Body weight did not significantly differ at any time points between the 2 groups. The present study is the first demonstrating rMETase efficacy in a PDOX model, suggesting potential clinical development, especially in recalcitrant cancers such as Ewing's sarcoma

Targeted therapy with a Salmonella typhimurium leucine-arginine auxotroph cures orthotopic human breast tumors in nude mice.

Abstract We report here a modified auxotrophic strain of Salmonella typhimurium that can target and cure breast tumors in orthotopic mouse models. We have previously reported development of a genetically modified strain of S. typhimurium, selected for prostate tumor targeting and therapy in vivo. The strain, termed S

Electrochemical synthesis of a ferrocenecarboxylic acid–C60 composite and its electrocatalysis to hydrogen peroxide

A new ferrocenecarboxylic acid–C60 composite (Fc–C60) has been synthesized by controlled potential electrolysis. A composite modified glassy carbon electrode has been prepared based on its good electrochemical activity. The modified electrode in 0.1 M NaClO4 solution shows a reversible oxidation wave at E1/2 = 0.32 V (vs. SCE) attributed to the oxidation of the ferrocene entity and a quasi-reversible reduction wave of C60 entity at E1/2 = −0.54 V (vs. SCE). Electrocatalytic studies show that Fc–C60 at the modified electrode can mediate the reduction of hydrogen peroxide (H2O2), and a broad linear range from 1.2 μM to 21.9 mM for H2O2 were obtained with a determination limit of 2.5 × 10−7 M by amperometry. Keywords: C60, Electrochemistry, Ferrocenecarboxylic acid, Electrocatalysis, Hydrogen peroxid