Reduced Malignancy of Super Methotrexate-resistant Osteosarcoma Cells With Dihydrofolate Reductase Amplification Despite Paradoxical Gain of Oncogenic PI3K/AKT/mTOR and c-MYC expression.

BACKGROUND/AIM: Methotrexate (MTX) resistance in osteosarcoma leads to a very poor prognosis. In the present study, in order to further understand the basis and ramifications of MTX resistance in osteosarcoma, we selected an osteosarcoma cell line that has a 5,500-fold-increased MTX IC50 Materials and Methods: The super MTX-resistant 143B osteosarcoma cells (143B-MTXSR) were selected from MTX-sensitive parental human 143B osteosarcoma cells (143B-P) by continuous culture with step-wise increased amounts of MTX. To compare the malignancy of 143B-MTXSR and 143B-P, colony-formation capacity was compared with clonogenic assays on plastic and in soft agar. In addition, tumor growth was compared with orthotopic xenograft mouse models of osteosarcoma. Expression of dihydrofolate reductase (DHFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), and myelocytomatosis oncogene (MYC) was examined with western immunoblotting and compared in 143B-MTXSR and 143B-P cells. RESULTS: 143B-MTXSR had a 5,500-fold increase in the MTX IC50 compared to the parental 143B-P cells. Expression of DHFR was increased 10-fold in 143B-MTXSR compared to 143B-P (p<0.01). 143B-MTXSR cells had reduced colony-formation capacity on plastic (p=0.032) and in soft agar (p<0.01) compared to 143B-P and reduced tumor growth in orthotopic xenograft mouse models (p<0.001). These results demonstrate that 143B-MTXSR had reduced malignancy. 143B-MTXSR also showed an increased expression of PI3K (p<0.01), phosphorylated (activated) AKT (p=0.031), phosphorylated mTOR (p=0.043), and c-MYC (p=0.024) compared to 143B-P. CONCLUSION: The present study demonstrates that the increased expression of DHFR, PI3K/AKT/mTOR and c-MYC appears to be linked to super MTX resistance and, paradoxically, to reduced malignancy. The present results suggest that DHFR may be a powerful tumor suppressor when highly amplified.

Hair-follicle associated pluripotent (HAP)-cell-sheet implantation enhanced wound healing in diabetic db/db mice.

Diabetes often results in chronic ulcers that fail to heal. Effective treatment for diabetic wounds has not been achieved, although stem-cell-treatment has shown promise. Hair-follicle-associated-pluripotent (HAP)-stem-cells from bulge area of mouse hair follicle have been shown to differentiate into keratinocytes, vascular endothelial cells, smooth muscle cells, and some other types of cells. In the present study, we developed HAP-cell-sheets to determine their effects on wound healing in type-2 diabetes mellitus (db/db) C57BL/6 mouse model. Flow cytometry analysis showed cytokeratin 15 expression in 64% of cells and macrophage expression in 3.6% of cells in HAP-cell-sheets. A scratch cell migration assay in vitro showed the ability of fibroblasts to migrate and proliferate was enhanced when co-cultured with HAP-cell-sheets. To investigate in vivo effects of the HAP-cell-sheets, they were implanted into 10 mm circular full-thickness resection wounds made on the back of db/db mice. Wound closure was facilitated in the implanted group until day 16. The thickness of epithelium and granulation tissue volume at day 7 were significantly increased by the implantation. CD68 positive area and TGF-ß1 positive area were significantly increased; meanwhile, iNOS positive area was reduced at day 7 in the HAP-cell-sheets implanted group. After 21 days, CD68 positive areas in the implanted group were reduced to under the control group level, and TGF-ß1 positive area had no difference between the two groups. These observations strongly suggest that the HAP-cell-sheets implantation is efficient to facilitate early macrophage activity and to suppress inflammation level. Using immuno-double-staining against CD34 and α-SMA, we found more vigorous angiogenesis in the implanted wound tissue. The present results suggest autologous HAP-cell-sheets can be used to heal refractory diabetic ulcers and have clinical promise.

A case of AA amyloidosis complicated by proliferating pilomatricoma and a review of the literature.

Although AA amyloidosis is primarily caused by inflammatory conditions, associations between AA amyloidosis and solid cancers have occasionally been described. Herein, we report the case of a 48-year-old man in whom resection of a proliferating pilomatricoma with deposition of AA amyloid resulted in remission of concomitant AA gastrointestinal amyloidosis. A rapidly growing, giant, reddish, ulcerated tumor measuring 16 × 13 cm in size was identified on the upper left arm on a visit to our hospital. Gastrointestinal AA amyloidosis was diagnosed from colorectal mucosal biopsy at the same time, and weight loss and profuse diarrhea were clinically evident. As treatment, the tumor was resected with a 10-mm surgical margin. Histologically, the tumor predominantly comprised a lobular proliferation of basophilic cells peripherally, filled with eosinophilic, cornified material and shadow cells with mitoses observed in basophilic cells. Specimens revealed eosinophilic, homogeneous deposits around tumor nests, which were confirmed as amyloid deposits by positive staining with Congo red stain. These deposits were immunohistochemically positive on staining with anti-serum amyloid A antibody. Collectively, proliferating pilomatricoma with AA amyloidosis was diagnosed. After tumor resection, chronic diarrhea resolved and no amyloid deposition was apparent in colorectal biopsy. It is important to remember that if amyloid deposition is present in a tumor, aggressive tumor excision may alleviate systemic amyloidosis.

Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells.

Methionine restriction of glioma does not induce MGMT and greatly improves temozolomide efficacy in an orthotopic nude-mouse model: A potential curable approach to a clinically-incurable disease.

Glioma is a highly recalcitrant disease with a 5-year survival of 6.8 %. Temozolomide (TMZ), first-line therapy for glioma, is more effective in O6-methylguanine-DNA methyltransferase (MGMT)-negative gliomas than in MGMT-positive gliomas as MGMT confers resistance to TMZ. Methionine restriction is effective for many cancers in mouse models including glioma. The concern is that methionine restriction could induce MGMT by decreasing DNA methylation and confer resistance to TMZ. In the present study, we investigated the efficacy of combining methionine restriction with TMZ for the treatment of MGMT-negative glioma, and whether methionine restriction induced MGMT. Human MGMT-negative U87 glioma cells were used to determine the efficacy of TMZ combined with methionine restriction. Recombinant methioninase (rMETase) inhibited U87 glioma growth without induction of MGMT in vitro. The combination of rMETase and TMZ inhibited U87 cell proliferation more than either agent alone in vitro. In the orthotopic nude-mouse model, the combination of TMZ and a methionine-deficient diet was much more effective than TMZ alone: two mice out of five were cured of glioma by the combination. No mice died during the treatment period. Methionine restriction enhanced the efficacy of TMZ in MGMT-negative glioma without inducing MGMT, demonstrating potential clinical promise for improved outcome of a currently incurable disease.

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.

The Combination of Methioninase and Ethionine Exploits Methionine Addiction to Selectively Eradicate Osteosarcoma Cells and Not Normal Cells and Synergistically Down-regulates the Expression of C-MYC.

BACKGROUND/AIM: The fundamental and general hallmark of cancer cells, methionine addiction, termed the Hoffman effect, is due to overuse of methionine for highly-increased transmethylation reactions. In the present study, we tested if the combination efficacy of recombinant methioninase (rMETase) and a methionine analogue, ethionine, could eradicate osteosarcoma cells and down-regulate the expression of c-MYC. MATERIALS AND METHODS: 143B osteosarcoma cells and Hs27 normal human fibroblasts were tested. The efficacy of rMETase alone and ethionine, alone and in their combination, on cell viability was determined with the WST-8 assay on 143B cells and Hs27 cells. c-MYC expression was examined with western immunoblotting and compared in 143B cells treated with/without rMETase, ethionine, or the combination of both rMETase and ethionine. RESULTS: 143B cells were more sensitive to both rMETase and ethionine than Hs 27 cells, with the following IC50s: rMETase (143B: 0.22 U/ml; Hs27: 0.82 U/ml); ethionine (143B: 0.24 mg/ml; Hs27: 0.42 mg/ml). The combination of rMETase and ethionine synergistically eradicated 143B cells, lowering the IC50 for ethionine 14-fold compared to ethionine alone (p<0.001). In contrast, Hs27 fibroblasts were relatively resistant to the combination. The expression of c-MYC was significantly down-regulated only by the combination of rMETase and ethionine in 143B cells (p<0.001). CONCLUSION: In the present study, we showed, for the first time, the synergistic combination efficacy of rMETase and ethionine on osteosarcoma cells in contrast to normal fibroblasts, which were relatively resistant. The combination of rMETase and ethionine down-regulated c-MYC expression in the cancer cells. The present results indicate the combination of rMETase and ethionine may reduce the malignancy of osteosarcoma cells and can be a potential future clinical strategy.

Targeting Autophagy With the Synergistic Combination of Chloroquine and Rapamycin as a Novel Effective Treatment for Well-differentiated Liposarcoma.

BACKGROUND/AIM: Liposarcoma is a type of soft-tissue sarcoma arising from fat tissue. It is relatively common among soft-tissue sarcomas. Chloroquine (CQ), an antimalarial drug, can inhibit autophagy and induce apoptosis in cancer cells. Rapamycin (RAPA) is an inhibitor of mTOR. The combination of RAPA and CQ is a strong inhibitor of autophagy. Previously, we showed that the combination of RAPA and CQ was effective against a de-differentiated liposarcoma patient-derived orthotopic xenograft (PDOX) mouse model. In the present study, we investigated the mechanism of efficacy of the combination of RAPA and CQ to target autophagy in a well-differentiated liposarcoma (WDLS) cell line in vitro. MATERIALS AND METHODS: The human WDLS cell line 93T449 was used. The WST-8 assay was used to test the cytotoxicity of RAPA and CQ. Western blotting was used to detect microtubule-associated protein light chain 3-II (LC3-II) which is a component of autophagosomes. Immunostaining of LC3-II was also performed for autophagosome analysis. Τhe TUNEL assay was used to detect apoptotic cells, and apoptosis-positive cells were counted in three randomly selected microscopic fields for statistical validation. RESULTS: RAPA alone and CQ alone inhibited the viability of 93T449 cells. The combination of RAPA and CQ inhibited 93T449 cell viability significantly more than either drug alone and increased the number of autophagosomes which led to extensive apoptosis. CONCLUSION: The combination of RAPA and CQ increased the number of autophagosomes which led to apoptosis in 93T449 WDLS cells, suggesting novel effective treatment for this recalcitrant cancer by targeting autophagy.

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

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

Meckel's Diverticulum Carcinoma Is Arrested by Oxaliplatinum and Eradicated by 5-Fluorouracil in a PDX Mouse Model Indicating Candidate First-line Treatment for a Rare Cancer.

BACKGROUND/AIM: Meckel's diverticulum carcinoma (MDCa) is extremely rare. It is often advanced when found, and the prognosis is poor. Effective treatment for this cancer has not yet been developed. We previously established a patient-derived xenograft (PDX) nude-mouse model of MDCa. In the present study, we investigated the efficacy of oxaliplatinum (L-OHP) and 5-fluorouracil (5-FU) on an MDCa PDX nude-mouse model. MATERIALS AND METHODS: PDX mouse models of MDCa were divided into three groups (five mice per group): untreated control; L-OHP-treated; and 5-FU-treated. Tumor volumes of the three groups were compared after 2 weeks. RESULTS: L-OHP arrested tumor growth (p=0.038) and 5-FU apparently eradicated the tumor (p=0.007). CONCLUSION: L-OHP and 5-FU are candidates for clinical first-line therapy of MDCa.

Precise and Facile Endotracheal Lung-tumor-implantation Mouse Model Visualized by GFP Expression.

BACKGROUND/AIM: In osteosarcoma, lung metastasis is a major cause of cancer-related death, as the 5-year survival rate for patients with metastases is approximately only 20-30%. To develop improved therapeutic strategies against lung-metastatic osteosarcoma, an experimental lung-tumor-implantation mouse model is needed for basic research. In the present study, we developed a precise and facile endotracheal lung-tumor-implantation technique. MATERIALS AND METHODS: For establishment of the lung-tumor-implantation mouse model of metastatic osteosarcoma, 5 mice were used. A 15-mm longitudinal incision was made in the center of the neck to expose the salivary glands. The salivary glands were then split, exposing the trachea covered by the sternohyoid muscles. The trachea was then clearly exposed by cutting the sternohyoid muscles longitudinally. A 22 G gavage needle was tilted slightly toward the left side of the mouse and inserted from the oral cavity into the bronchus, with confirmation of the position of the tip of the gavage needle visualized through the tracheal wall, followed by injection of 0.5% crystal violet to first confirm the accuracy of endotracheal injection in the lung. A 143B-GFP cell suspension (2.0×106 cells/50 µl PBS) was then injected endotracheally in other mice. RESULTS: The procedure, including anesthesia and suturing, took approximately 10 minutes. The left lobe of the lung, in which crystal violet was injected endotracheally, was stained in 3 out of 3 mice (100%). 143B-GFP-osteosarcoma tumors were detected with GFP fluorescence in the left lobe of the lung in 3 out of 4 mice (75%), 5 weeks after endotracheal injection. One mouse died 4 weeks after 143B-GFP-cell implantation. CONCLUSION: This novel technique of establishing tumors in the lung via endotracheal injection of cancer cells is precise and facile and can be used widely, since neither a surgical microscope nor X-ray imaging are needed.

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

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

Oncogenes and Methionine Addiction of Cancer: Role of c-MYC.

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

Superiority of [11C]methionine over [18F]deoxyglucose for PET Imaging of Multiple Cancer Types Due to the Methionine Addiction of Cancer.

Positron emission tomography (PET) is widely used to detect cancers. The usual isotope for PET imaging of cancer is [18F]deoxyglucose. The premise of using [18F]deoxyglucose is that cancers are addicted to glucose (The Warburg effect). However, cancers are more severely addicted to methionine (The Hoffman effect). [11C]methionine PET (MET-PET) has been effectively used for the detection of glioblastoma and other cancers in the brain, and in comparison, MET-PET has been shown to be more sensitive and accurate than [18F]deoxyglucose PET (FDG-PET). However, MET-PET has been limited to cancers in the brain. The present report describes the first applications of MET-PET to cancers of multiple organs, including rectal, bladder, lung, and kidney. The results in each case show that MET-PET is superior to FDG-PET due to the methionine addiction of cancer and suggest that the broad application of MET-PET should be undertaken for cancer detection.

Direct implantation of hair-follicle-associated pluripotent (HAP) stem cells repairs intracerebral hemorrhage and reduces neuroinflammation in mouse model.

Intracerebral hemorrhage (ICH) is a leading cause of mortality with ineffective treatment. Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into neurons, glial cells and many other types of cells. HAP stem cells have been shown to repair peripheral-nerve and spinal-cord injury in mouse models. In the present study, HAP stem cells from C57BL/6J mice were implanted into the injured brain of C57BL/6J or nude mice with induced ICH. After allo transplantation, HAP stem cells differentiated to neurons, astrocytes, oligodendrocytes, and microglia in the ICH site of nude mice. After autologous transplantation in C57BL/6J mice, HAP stem cells suppressed astrocyte and microglia infiltration in the injured brain. The mRNA expression levels of IL-10 and TGF-ß1, measured by quantitative Real-Time RT-PCR, in the brain of C57BL/6J mice with ICH was increased by HAP-stem-cell implantation compared to the non-implanted mice. Quantitative sensorimotor function analysis, with modified limb-placing test and the cylinder test, demonstrated a significant functional improvement in the HAP-stem-cell-implanted C57BL/6J mice, compared to non-implanted mice. HAP stem cells have critical advantages over induced pluripotent stem cells, embryonic stem cells as they do not develop tumors, are autologous, and do not require genetic manipulation. The present study demonstrates future clinical potential of HAP-stem-cell repair of ICH, currently a recalcitrant disease.

Hair follicle associated pluripotent (HAP) stem cells jump from transplanted whiskers to pelage follicles and stimulate hair growth.

Stimulation of hair growth in hair loss has been a difficult goal to achieve. Hair follicle-associated pluripotent (HAP) stem cells express nestin and have been shown to differentiate to multiple cell types including keratinocytes, neurons, beating cardiac muscles and numerous other cell types. HAP stem cells originate in the bulge area of the hair follicle and have been shown to migrate within and outside the hair follicle. In the present study, the upper part of vibrissa follicles from nestin-driven green-fluorescent protein (GFP) transgenic mice, containing GFP-expressing HAP stem cells, were transplanted in the dorsal area of athymic nude mice. Fluorescence microscopy and immunostaining showed the transplanted HAP stem cells jumped and targeted the bulge and hair bulb and other areas of the resident nude mouse pelage follicles where they differentiated to keratinocytes. These results indicate that transplanted nestin-GFP expressing HAP stem cells jumped from the upper part of the whisker follicles and targeted nude-mouse hair follicles, which are genetically deficient to grow normal hair shafts, and differentiated to keratinocytes to produce normal mature hair shafts. The resident nude-mouse pelage follicles targeted by jumping whisker HAP stem cells produced long hair shafts from numerous hair follicles for least 2 hair cycles during 36 days, demonstrations that HAP stem cells can stimulate hair growth. The present results for hair loss therapy are discussed.

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

BACKGROUND/AIM: Invasive lobular carcinoma (ILC) of the breast has a low complete-response rate in the neoadjuvant-chemotherapy setting. The addiction to methionine is a fundamental and ubiquitous characteristic of cancer cells, termed the Hoffman effect. We have previously targeted methionine addiction of breast cancer with recombinant methioninase (rMETase) using patient-derived orthotopic xenograft (PDOX) models. The aim of the present study was to determine the efficacy of methionine restriction with rMETase and a low-methionine diet combined with first-line neo-adjuvant chemotherapy, in a patient with metastatic ILC of the breast. CASE REPORT: A 62-year-old female was diagnosed with metastatic ipsilateral axillary-lymph-node recurrence of breast ILC 3 years after mastectomy. The patient underwent [11C]-methionine positron-emission tomography (METPET) which showed extensive methionine accumulation in the ipsilateral axillary lymph nodes, indicating the presence of cancer cells. The patient received standard neo-adjuvant chemotherapy, which consisted of 3 months of doxorubicin and cyclophosphamide followed by 3 months of docetaxel from March 2022. The patient also went on a low-methionine diet and daily oral rMETase as a supplement every 6 hours concurrently with six months chemotherapy. The patient's blood carcinoembryonic antigen (CEA) level decreased gradually, and computed tomography findings showed loss of axillary lymph-node metastases in the first 3 months of neo-adjuvant chemotherapy with doxorubicin and cyclophosphamide combined with rMETase and a low-methionine diet. A complete response was demonstrated by METPET at 6 months, at conclusion of docetaxel chemotherapy. CONCLUSION: Combination therapy of doxorubicin and cyclophosphamide followed by docetaxel combined with methionine restriction led to a remarkable complete response that is expected in fewer than 10% of patients with ILC of the breast treated with neo-adjuvant chemotherapy alone. The present results suggest that methionine restriction in combination with doxorubicin and cyclophosphamide followed by docetaxel may be effective, after METPET has demonstrated the presence of methionine-addicted axillary-lymph-node metastases in ILC of the breast.

Reversion of methionine addiction of osteosarcoma cells to methionine independence results in loss of malignancy, modulation of the epithelial-mesenchymal phenotype and alteration of histone-H3 lysine-methylation.

Methionine addiction, a fundamental and general hallmark of cancer, known as the Hoffman Effect, is due to altered use of methionine for increased and aberrant transmethylation reactions. However, the linkage of methionine addiction and malignancy of cancer cells is incompletely understood. An isogenic pair of methionine-addicted parental osteosarcoma cells and their rare methionine-independent revertant cells enabled us to compare them for malignancy, their epithelial-mesenchymal phenotype, and pattern of histone-H3 lysine-methylation. Methionine-independent revertant 143B osteosarcoma cells (143B-R) were selected from methionine-addicted parental cells (143B-P) by their chronic growth in low-methionine culture medium for 4 passages, which was depleted of methionine by recombinant methioninase (rMETase). Cell-migration capacity was compared with a wound-healing assay and invasion capability was compared with a transwell assay in 143B-P and 143B-R cells in vitro. Tumor growth and metastatic potential were compared after orthotopic cell-injection into the tibia bone of nude mice in vivo. Epithelial-mesenchymal phenotypic expression and the status of H3 lysine-methylation were determined with western immunoblotting. 143B-P cells had an IC50 of 0.20 U/ml and 143B-R cells had an IC50 of 0.68 U/ml for treatment with rMETase, demonstrating that 143B-R cells had regained the ability to grow in low methionine conditions. 143B-R cells had reduced cell migration and invasion capability in vitro, formed much smaller tumors than 143B-P cells and lost metastatic potential in vivo, indicating loss of malignancy in 143B-R cells. 143B-R cells showed gain of the epithelial marker, ZO-1 and loss of mesenchymal markers, vimentin, Snail, and Slug and, an increase of histone H3K9me3 and H3K27me3 methylation and a decrease of H3K4me3, H3K36me3, and H3K79me3 methylation, along with their loss of malignancy. These results suggest that shifting the balance in histone methylases might be a way to decrease the malignant potential of cells. The present results demonstrate the rationale to target methionine addiction for improved sarcoma therapy.

Oral-recombinant Methioninase in Combination With Rapamycin Eradicates Osteosarcoma of the Breast in a Patient-derived Orthotopic Xenograft Mouse Model.

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

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

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