The limited ability of current treatments to control metastasis and the proposed antitumor properties of specific nutrients prompted us to examine the effect of a specific formulation (nutrient ...supplement NS) of lysine, proline, arginine, ascorbic acid, and green tea extract in vivo on the development of N-methyl-N-nitrosourea (MNU)-induced mammary tumors in rats.
A single intraperitoneal dose of MNU was injected into each of 20 female Sprague-Dawley rats (aged 50 days) to induce tumors. Two weeks after MNU treatment, a time by which the animals had recovered from MNU-induced toxicity, the rats were divided into two groups. Rats in group 1 (n = 10) were fed Purina chow diet, whereas those in group 2 (n = 10) were fed the same diet supplemented with 0.5% NS. After a further 24 weeks, the rats were killed and tumors were excised and processed.
NS reduced the incidence of MNU-induced mammary tumors and the number of tumors by 68.4%, and the tumor burden by 60.5%. The inhibitory effect of NS was also reflected by decreased tumor weight; the tumor weights per rat and per group were decreased by 41% and 78%, respectively. In addition, 30% of the control rats developed ulcerated tumors, in contrast to 10% in the nutrient supplemented rats.
These findings suggest that the specific formulation of lysine, proline, arginine, ascorbic acid, and green tea extract tested significantly reduces the incidence and growth of MNU-induced mammary tumors, and therefore has strong potential as a useful therapeutic regimen for inhibiting breast cancer development.
Adult sarcomas are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the ECM and basement membrane, allowing cancer ...cells to spread to distal organs. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). Our main objective was to study the effect of a nutrient mixture (NM) on the activity of u-PA, MMPs and TIMPs in various human adult sarcomas. Human fibrosarcoma (HT-1080), chondrosarcoma (SW-1353), liposarcoma (SW-872), synovial sarcoma (SW-982) and uterine leimyosarcoma (SK-UT-1) cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1,000 μg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Fibrosarcoma, chondrosarcoma, liposarcoma and leiomyosarcoma cancer cell lines expressed u-PA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to u-PA were detected for synovial sarcoma cells. On gelatinase zymography, fibrosarcoma, chondrosarcoma, liposarcoma and synovial sarcoma showed bands corresponding to MMP-2 and MMP-9 with enhancement of MMP-9 with PMA (100 ng/ml) treatment. Uterine leiomyosarcoma showed strong bands corresponding to inactive and active MMP-9 and a faint band corresponding to MMP-9 dimer induced with PMA treatment, but no MMP-2 band. NM inhibited their expression in a dose-dependent manner. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in treatment of adult sarcomas.
Adriamycin (ADR), an antineoplastic antibiotic used in cancer therapy, is associated with toxicity to vital organs with long-term use. A nutrient mixture (NM) has previously been shown to exhibit a ...broad spectrum of therapeutic properties. The aim of the present study was to determine whether the NM is useful for preventing ADR-induced hepatic and nephric toxicity. Six-week-old male BALB/c mice were divided into four groups of six animals each. Groups A and C were fed a regular diet for three weeks and groups B and D were fed a diet supplemented with 1% NM. After three weeks, the mice in groups C and D received 20 mg/kg body weight ADR intraperitoneally, while those in groups A and B received saline alone. Animals were sacrificed after 24 h, blood samples were collected and serum was obtained for clinical chemistry. Organs were also excised and weighed. Administration of ADR to group C (control diet) resulted in a marked increase in hepatic alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transferase levels and renal blood urea nitrogen, creatinine and uric acid serum markers. However, in group D (NM 1% diet), the serum markers were comparable with the levels of group A and B. Therefore, the results indicate that NM has the potential to protect against ADR-induced hepatic and nephric damage.
Lung cancer, the most prevalent cancer worldwide and malignant mesothelioma are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretion. ...Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPS). Our main objective was to study the effect of a nutrient mixture (NM) on the activity of u-PA, MMPs and TIMPs on human lung and malignant mesothelioma (MM) cell lines. Human lung cancer (A-549 and Calu-3) and malignant mesothelioma (MSTO-211H) cell lines were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 μg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Both lung cancer cell lines expressed u-PA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to u-PA were detected for the MSTO-211H MM cell line. On gelatinase zymography, A-549 cells showed one band corresponding to MMP-2 and induction of MMP-9 with PMA (100 ng/ml) treatment. MSTO-211H showed two bands, an intense band corresponding to MMP-2 and a faint band corresponding to MMP-9; MMP-9 was enhanced significantly with PMA treatment. NM inhibited their expression in both cell lines in a dose-dependent manner. Calu-3 showed no MMP-2 or MMP-9 expression. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in the treatment of lung and mesothelioma cancers.
Cervical cancer is one of the most commonly diagnosed cancers and a significant cause of mortality in women worldwide. Although cervical cancer is fully treatable in the early stages, once it has ...metastasized, patient outcome is poor. The objective of the present study was to investigate the effect of dietary supplementation with a nutrient mixture (NM) containing lysine, ascorbic acid, proline, green tea extract and other micronutrients on the expression of extracellular matrix (ECM) proteins in HeLa cell xenografts in nude female mice. After housing for 1 week, female athymic nude mice between 5 and 6 weeks of age (n=12) were inoculated subcutaneously with 3×106 HeLa cells in phosphate-buffered saline and Matrigel and randomly divided into two groups. These were the control group, in which the mice were fed with regular mouse chow, and the NM group, in which the mice were fed with the regular diet supplemented with 0.5% NM (w/w). After 4 weeks, the tumors were excised and processed for histology. Tumor growth was evaluated and the tumors were stained for the ECM proteins collagen I, collagen IV, fibronectin, laminin, periodic acid-Schiff (PAS) and elastin. NM strongly inhibited (by 59%, P=0.001) the growth of HeLa xenografts in nude mice. Tumors from control mice exhibited little to no collagen I expression either internally or in the fibrous capsule, while tumors from the NM group expressed collagen I in the fibrous capsule and within the tumor. Tumors from the control group showed diffuse cytoplasmic and capsular collagen IV with abundant nucleated cells. NM treatment substantially increased collagen IV production and induced a dense fibrous network of collagen IV with chambers that surrounded live nucleated cells and large amounts of necrotic cell debris. Tumors from the mice fed with the NM exhibited a well-defined border of fibronectin in the capsule and intense areas of staining internally whereas control group tumors showed less overall fibronectin with sporadic internal staining and little in the fibrous capsule. Although laminin appeared abundantly in control and NM-treated tumors, the NM group tumors exhibited a chamber-like network of laminin internally. Tumors from the control group exhibited internal areas of intense PAS staining, whereas tumors from the NM-treated group exhibited a more uniform diffuse pattern of PAS staining. In conclusion, NM supplementation of HeLa xenograft-bearing female nude mice demonstrated a potent inhibition of tumor growth and enhancement of ECM proteins, suggesting the therapeutic value of this specific nutrient complex in the treatment of cervical cancer.
OBJECTIVE: The prevention and treatment of cancer remain a challenge. Current treatments are largely unsuccessful due to high toxicity. The most effective way to reduce global mortality from cancer ...is to block the initial stages of the disease, common to all types of cancer – invasion and metastasis. The elevated levels of matrix metalloproteinases, such as MMP-9 play a key role in tumorigenesis, angiogenesis, apoptosis, cancer invasion and metastasis. Among various therapeutic modalities, vaccines are the most effective and affordable approaches against diseases in general. In the global fight against cancer, a vaccine capable to impede MMP-2 and MMP-9 activity could open the door for effective prevention -- and even cure. We previously reported that mice immunized with synthetic oligopeptides containing specific amino acid sequences from human MMP-2 and MMP-9 showed a significant reduction in melanoma tumors and tumor burden. MATERIALS AND METHODS: Here, we tested a syngeneic approach to cancer vaccines by investigating whether immunization of mice with rodent derived MMP-9 oligopeptides would generate sufficient immune response and anticancer efficacy. Accordingly, C57Bl/6 mice were immunized with three oligopeptides containing specific sequences from rat MMP-9 and two oligopeptides from mouse MMP-9. All these peptides showed to be highly immunogenic in mice. RESULTS: Subsequently, the immunized mice challenged with B16FO melanoma cells developed significantly smaller tumors and had reduced tumor burden. The weight gain in vaccinated and control mice was comparable. In addition, no significant differences were observed in serum clinical chemistry, hematological parameters and the histopathology of major organs in relation to test peptides in the immunized mice. CONCLUSIONS: Our findings confirm that MMP peptide-based vaccines can be a viable strategy for cancer therapy.
Retinoblastoma is one of the most common ocular malignancies in children under the age of six. Occasionally, retinoblastoma metastasizes to extraocular organs including the bone, lung and brain. Left ...untreated, retinoblastoma is fatal. At present, there is no effective treatment for metastatic retinoblastoma. We investigated the antineoplastic activity of a nutrient mixture (NM) (lysine, proline, ascorbic acid and green tea extract) at concentrations of 10, 50, 100, 500 and 1,000 μg/ml in triplicate at each dose in the human malignant retinoblastoma Y-79 cell line. The parameters used were cell proliferation, expression of matrix metalloproteinases (MMPs), invasion through Matrigel, morphology and apoptosis. Cell viability was assessed by trypan blue dye exclusion test. Invasion was evaluated through Matrigel and MMP activity by gelatinase zymography. H&E staining for morphological cell alterations and apoptotic studies using the Live Green Poly Caspase Detection kit were also conducted. The nutrient mixture at 10-100 μg/ml demonstrated approximately 25% toxicity towards Y-79 retinoblastoma cells and significant toxicity at 500 and 1,000 μg/ml. The Y-79 cells secreted only MMP-2 as demonstrated by zymography; the nutrient mixture had no effect on MMP-2 expression up to 100 μg/ml, but completely blocked it at 500 μg/ml. Importantly, Y-79 retinoblastoma cells were not invasive through Matrigel. H&E staining showed cell morphological changes related to apoptosis, which was confirmed using the Live Green Poly Caspase Detection kit. Our results suggest that this nutrient mixture, which inhibited cell proliferation, expression of MMP-2 and induced apoptosis, may be a candidate for further exploration for its therapeutic potential in metastatic retinoblastoma.
Amiodarone (Amio), a potent anti-arrhythmic drug, is associated with life-threatening pulmonary toxicity involving fibroses and inflammation. A unique nutrient mixture (NM) consisting of lysine, ...proline, ascorbic acid, N-acetyl cysteine and green tea extract has previously been shown to exhibit a broad spectrum of pharmacological, therapeutic, cardiovascular and chemopreventive properties. The present study was undertaken to determine whether the NM exhibits preventive effects on Amio-induced cardiac toxicity. Six-week-old male BALB/c mice were divided into four groups (A-D) of six animals per group. Mice in groups A and C were fed a regular diet for three weeks, while the diets of the mice in groups B and D were supplemented with 1% NM during that period. After three weeks, the mice in groups C and D received daily Amio injections of 50 mg/kg body weight intraperitoneally for 4 days, whilst those in groups A and B received saline alone. At 24 h after the final dose, mice were sacrificed, blood was withdrawn and serum was collected for clinical chemistry of the heart enzymes creatine phosphokinase (CPK) and aspartate aminotransferase (AST). In addition, livers, kidneys, hearts and lungs were excised and weighed. No significant differences in weight gain were identified among the groups and liver, kidney, heart and lung weights were comparable in all four groups. Administration of Amio to group C resulted in a significant increase in serum CPK levels, whereas in NM-fed group D, the CPK levels were comparable to those in the saline injection groups, A and B. Amio administration also resulted in a significant increase in serum AST levels in group C, but not in the group D animals which exhibited similar levels to those of groups A and B. Therefore, the results indicate that NM has the potential to protect against Amio-induced cardiac toxicity.
Neuroblastoma, a peripheral nervous system cancer that can be highly invasive and metastatic, accounts for 8-10% of all solid childhood tumors in children under the age of 15 years. Despite multiple ...clinical efforts, prognosis remains poor for this enigmatic disease. A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has shown significant antitumor effects. Using the nude mouse xenograft model, we investigated the efficacy of NM. We also tested the effect of NM in vitro, evaluating cell viability, secretion of the matrix metalloproteinases (MMP)-2 and MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 secretion, Matrigel invasion and cellular apoptosis and morphology. Athymic nude mice 5-6 weeks of age were inoculated with 3×106 SK-N-MC neuroblastoma cells subcutaneously and randomly divided into two groups. Group A was fed a regular diet and group B a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histology. We also tested the effect of NM in vitro. NM inhibited the growth of xenograft tumors by 22% (P=0.04); and, in vitro, NM induced dose-dependent inhibition of cell proliferation with a decrease of 27% (P=0.001) and 36% (P=0.002) at 500 and 1000 μg/ml NM compared to the control, respectively. Zymography revealed MMP-2 secretion in normal cells and PMA (100 ng/ml)-induced MMP-9 secretion. NM inhibited the secretion of both MMPs with total blockage at a concentration of 100 μg/ml. Reverse zymography demonstrated a dose-dependent increase in TIMP-2 expression by NM. Notable, SK-N-MC human neuroblastoma cells were not invasive through Matrigel. NM induced dose-dependent apoptosis of SK-N-MC cells. The results suggest that NM may have therapeutic potential in treating neuroblastoma.
Abstract
Current cancer therapies are based on chemotherapy and radiation. These therapeutic approaches can initially reduce the tumor mass or tumor burden by killing tumor cells. It has been ...postulated that the resulting dying tumor cells, or tumor debris, can act as sources of tumor stimulation on surviving cells leading to reappearance of the cancer, and thereby drastically reducing the survival rate. In our study we investigated whether 4T1 debris induced by chemotherapeutic agent docetaxel would affect breast tumor growth in female nude mice. Female athymic nude mice were divided into three groups: Group 1 was inoculated with 4T1 cells; Group 2 with 4T1 cells together with their debris and Group 3 with debris alone. All mice were fed a regular diet. After four weeks, the mice were sacrificed; tumors were excised, weighed and processed for histology and immunohistochemistry for inflammation parameters. Tumor weight significantly increased in Group 2 mice compared to the control Group 1. Group 3 mice developed no tumors. Although the tumor histology for both Groups 1 and 2 were similar, there were significant differences in inflammatory parameters in the two groups. Tumors developed in Group 2 mice had intense staining patterns for TNF-alpha, IL-6, Ki-67, i-Nos and VEGF compared to tumors in the control Group 1. Our results show that dying cells from docetaxel treatment, or tumor debris, had stimulatory effect on growth of breast cancer 4T1 cells in nude mice which was associated with increased inflammation. This study represents a novel method to evaluate the effects of chemotherapy or radiation on surviving tumor cells and to investigate the effect of various inhibitors on the growth recurrence.
Citation Format: M Waheed Roomi, Aleksandra Niedzwiecki, Matthias Rath. Dying tumor cells (tumor debris) stimulate the growth of 4T1 breast cancer tumor in female athymic nude mice abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2172.
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