Strong clinical and experimental evidence demonstrates association of elevated levels of matrix metalloproteinase MMP-9 with cancer progression, metastasis and shortened patient survival, as it plays ...a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. MMP-9 is secreted in both the monomeric and dimeric form. Although there is little research on MMP-9 dimers, some studies have shown the dimer to be associated with more aggressive tumor progression. Our objective was to study the relative secretion patterns of MMP-9 monomer and dimer in a variety of cancer cell lines and the effect of a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract on MMP-9 secretion. The cancer cell lines were grown in their respective media, supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with NM at 0,10, 50, 100, 500 and 1000 µg/ml. Parallel sets of cultures were treated with PMA (100 ng/ml) for induction of MMP-9. Cell MMP-9 secretion was assayed by gelatinase zymography. MMP-9 dimer secretion patterns of cancer cells fell into different categories. We observed no MMP-9 dimer in prostate DU-145 and PC-3, pancreatic MIA-Pa-Ca2, colon HCT-116, bladder T-24, head and neck FaDu, glioblastoma A-172, T-98 and LN-18 and leukemia HL-60, Jurkat, and Raji cell lines. MMP-dimer secretion only with PMA induction was seen in breast MCF-7 and MDA-MB-231, uterine SK-UT-1, lung A-549, tongue SC-25, melanoma A2058, osteosarcoma U-2OS, rhabdomyosarcoma, fibrosarcoma HT-1080, chondrosarcoma SW-1350 and liposarcoma SW-872. Cervical HeLa and DoTc 2 4510, renal 786-0 and HCC SK-Hep-1 cells exhibited MMP-9 dimer without PMA treatment and increased secretion with PMA treatment. Sarcomas had the highest levels of MMP-9 monomer and dimer with and without PMA among these cancer cell lines. Cervical, uterine and male breast cancer cell lines showed the next highest levels of MMP-9, followed by breast cancer cell lines. Melanoma, renal, lung, head and neck and HCC showed lower levels and prostate, glioblastoma, bladder and leukemia cell lines the lowest. NM showed dose-dependent inhibition of MMP-9 monomer and dimer in all cell lines tested. In conclusion, high MMP-9 and dimer secretion levels correlated with the most aggressive cancer cell lines. NM was effective in inhibiting MMP-9 and dimer secretion in all cell lines tested, suggesting its therapeutic potential as an antimetastatic agent.
Type IV collagenase matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, have been found to promote invasion and metastasis of malignant tumors. Extracellular matrix (ECM) degradation by ...MMPs and increased expression of MMPs in cancer cells and tumor microvascular endothelial cells make MMPs an attractive target for cancer. Focused on a common pathomechanism of cancer growth and invasion, the disintegration of connective tissue, we used natural approaches to increase the integrity and strength of connective tissues. Utilizing the principle of nutrition synergy, we developed a novel micronutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract. This study evaluates the potency of the components EGCG and green tea extract independently compared to that of NM on modulation of patterns of MMP-2 and MMP-9 expression in four cancer cell lines expressing MMP-2, MMP-9 or both. Human fibrosarcoma (HT-1080), hepatocellular carcinoma (SK-Hep-1), glioblastoma (T-98G), uterine leiomyosarcoma (SK-UT-1) cell lines were obtained from ATCC and grown in minimum essential medium (MEM) supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with agents dissolved in media and tested at concentrations indicated in triplicate at each dose. Cells were also treated with PMA 100 ng/ml to study enhanced expression of MMP-9. MMP expression was assessed by gelatinase zymography. Fibrosarcoma and hepatocellular carcinoma cells expressed both MMP-2 and MMP-9. Glioblastoma cells expressed MMP-2 and PMA treatment induced MMP-9 expression. Uterine leimyosarcoma cells expressed no MMPs but PMA induced MMP-9. NM was the most potent dose-dependent inhibitor of MMPs, followed by green tea extract and EGCG. In conclusion, these results suggest the enhanced efficacy of nutrients working in synergy to modulate complex pathways such as MMP expression.
Head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukemia are the major causes of mortality and morbidity in Fanconi anemia (FA) patients. The objective of this study was to ...investigate the antineoplastic activity of PB, an antineoplastic nutrient mixture (containing quercetin, curcumin, green tea, cruciferex and resveratrol) on human FA HNSCC in vitro and in vivo. Human FA HNSCC cell line OHSU-974 (Fanconi Anemia Research Fund) was cultured in RPMI medium supplemented with 20% FBS and anti-biotics. At near confluence, cells were treated in triplicate with different concentrations of PB: 0, 10, 25, 50, 75 and 100 µg/ml. Cells were also treated with PMA to induce MMP-9 activity. Cell proliferation was detected by MTT assay, secretion of MMPs by gelatinase zymography, invasion through Matrigel, migration by scratch test and morphology by hematoxylin and eosin (H&E) staining. In vivo, athymic male nude mice (n=12) were inoculated with 3x106 OHSU-974 cells subcutaneously and randomly divided into two groups: group A was fed a regular diet and group B a regular diet supplemented with 1% PB. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histology. NM inhibited the growth of OHSU-974 tumor by 67.6% (p<0.0001) and tumor burden by 63.6% (p<0.0001). PB demonstrated dose-dependent inhibition of cell proliferation, with 27% (p=0.0003) and 48% (p=0.0004) toxicity at 75 and 100 µg/ml, respectively. Zymography revealed MMP-2 and PMA-induced MMP-9 secretion. PB suppressed secretion of both MMPs in a dose-dependent manner, with total block of both at 50 µg/ml. PB inhibited cell migration (by scratch test) and OHSU-974 invasion through Matrigel in a dose-dependent fashion with total block at 50 µg/ml. H&E staining showed no morphological changes below 50 µg/ml. The results suggest that PB has potential therapeutic use in the treatment of human FA HNSCC.
We investigate the influence of the geomagnetic field (GF) on the Imaging Air Cherenkov Telescope technique for two northern (Tenerife and San Pedro Martir) and three southern (Salta, Leoncito and ...Namibia (the H.E.S.S.-site)) site candidates for Cherenkov Telescope Array (CTA) observatories. We use the CORSIKA and sim_telarray programs for Monte Carlo simulations of gamma ray showers, hadronic background and the telescope response. We focus here on gamma ray measurements in the low energy, sub-100GeV, range. Therefore, we only consider the performance of arrays of several large telescopes. Neglecting the GF effect, we find (in agreement with previous studies) that such arrays have lower energy thresholds, and larger collection areas below 30GeV, when located at higher altitudes. We point out, however, that in the considered ranges of altitudes and magnetic field intensities, 1800–3600m a.s.l. and 0–40μT, respectively, the GF effect has a similar magnitude to this altitude effect. We provide the trigger-level performance parameters of the observatory affected by the GF effect, in particular the collection areas, detection rates and the energy thresholds for all five locations, which information may be useful in the selection of sites for CTA. We also find simple scaling of these parameters with the magnetic field strength, which can be used to assess the magnitude of the GF effect for other sites; in this work we use them to estimate the performance parameters for five sites: South Africa-Beaufort West, USA-Yavapai Ranch, Namibia-Calapanzi, Chile-La Silla and India-Hanle. We roughly investigate the impact of the geophysical conditions on gamma/hadron separation procedures involving image shape and direction cuts. We note that the change of altitude has an opposite effect at the trigger and analysis levels, i.e. gains in triggering efficiency at higher altitudes are partially balanced by losses in the separation efficiency. In turn, a stronger GF spoils both the shape and the direction discrimination of gamma rays, thus its effects at the trigger and analysis levels add up resulting in a significant reduction of the observatory performance. Overall, our results indicate that the local GF strength at a site can be equally important as its altitude for the low-energy performance of CTA.
Hemangiomas are the most common congenital vascular and benign tumor in infants and children. Most hemangiomas do not cause major symptoms to require intervention, however, the larger hemangiomas ...have tendency to bleed and may require surgical removal. Experimental studies have demonstrated the role of urokinase plasminogen activator (u-PA), especially cell surface u-PA, as an initiator of extra-cellular matrix proteolysis and associated tumor cell invasion.
To examine, whether the antitumor effects of a specific nutrient mixture are due to induction of apoptosis by inhibition of u-PA.
A nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract which has showed anticancer activity against a number of cancer cell lines was used as an experimental composition. EOMA cells were grown in appropriate media with antibiotics in 24-well tissue culture plates. At near confluence, the cells were treated with nutrition mixture at 10, 100, 1000 µg/ml in triplicate. Analysis of u-PA activity was carried out by fibrin zymography. Morphological changes and caspase activation associated with apoptosis induction was checked by H&E staining and Live Green caspase assay, respectively. Apoptosis inducing anticancer drug camptothecin (10 µM) was used as positive control.
The nutrition mixture exhibited dose response toxicity with maximum toxicity 55% (p < 0.001) at 1000 µg/ml. EOMA cells expressed u-PA, which was inhibited by nutrition mixture in a dose-dependent manner. The caspase analysis revealed a dose dependent increase in apoptosis of EOMA hemangioma cells, with an increasing apoptosis observed at 100 μg/ml, and maximum at 1000 μg/ml. Cells treated with nutrition mixture showed significantly more apoptotic changes than the control or camptothecin-treated cells.
These results suggest that NM may induce apoptosis of hemangioma cells in vitro thus warranting further investigation.
Objective: Therapeutic approach to cancer based on a vaccine targeting various types of cancer compared to being cancer-type specific has many advantages and can be applied to large population. Our ...previous in vivo studies demonstrated that a vaccine aimed at curbing cancer metastasis by inhibiting matrix metalloproteinases (MMPs) – Matrix Metalloproteinase -2 (MMP-2) and Matrix Metalloproteinase -9 (MMP-9) - has potential in preventing cancer. This study investigated mechanical aspects of anti-MMP-9 antibodies with previously documented anti-cancer efficacy in vivo, for inhibition of cancer cell migration and their binding efficacy. Materials and Methods: The antibodies against mouse and rat MMP-9 oligopeptides were isolated and their effects on cancer cell invasion through Matrigel were tested individually and in combinations. Binding to the corresponding MMP sequences was evaluated by Western blot. Results: All tested antibodies inhibited migration of both murine and human cancer cell lines: Human Prostate Cancer (DU145), Mouse Breast cancer (4T1) and Human Pancreas Carcinoma (MIA PaCa-2). The combination of these antibodies had enhanced effect on inhibiting cancer cells invasion. Antibody binding to MMP-9 was shown for all but one of the tested antibodies by Western Blot. Conclusions: Controlling matrix metalloproteinases (MMPs) is a powerful tool against metastasis - the most life-threatening aspect in all cancers. The in vitro efficacy of specific antibodies effective against MMP-9 was elucidated in this study. We showed that specific anti-MMP-9 antibodies inhibit extracellular matrix invasion of different types of cancer cells and confirmed their binding to corresponding MMP-9 peptides. Enhanced efficacy of these immunogenic peptides was observed when used in a combination. These results urge further work in this direction with its goal of developing a universal anti-cancer vaccine.
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.
Long-term survival of patients with breast cancer remains poor, due to metastasis and recurrence. We investigated the effects of a novel nutrient mixture (NM) containing ascorbic acid, lysine, ...proline and green tea extract in vitro and in vivo on 4T1 murine breast cancer, a representative model for metastatic breast cancer. After one week of isolation, 5-6-week-old female Balb/C mice were inoculated with 5x10⁵ 4T1 cells into the mammary pad and randomly divided into two groups; the control group was fed a regular diet and the NM group a regular diet supplemented with 0.5% NM. After four weeks, the mice were sacrificed and their tumors, lungs, livers, kidneys, hearts and spleens were excised and processed for histology. Dimensions (length and width) of tumors were measured using a digital caliper, and the tumor burden was calculated using the following formula: 0.5 x length x width. We also tested the effect of NM in vitro on 4T1 cells, measuring cell proliferation by MTT assay, MMP secretion by zymography, invasion through Matrigel, migration by scratch test and morphology by H&E staining. NM inhibited tumor weight and burden of 4T1 tumors by 50% (p=0.02) and 53.4% (p≤0.0001), respectively. Lung metastasis was profoundly inhibited by NM supplementation: mean number of colonies was reduced by 87% (p<0.0001) and mean weight of lungs by 60% (p=0.0001) compared to control mice. Metastasis to liver, spleen, kidney and heart was significantly reduced with NM supplementation. In vitro, NM exhibited 50% toxicity over the control at 250 and 500 µg/ml concentrations. Zymography demonstrated MMP-2 and MMP-9 secretion which was inhibited by NM in a dose-dependent manner, with virtual total inhibition of both at 1,000 µg/ml. Migration by scratch test and invasion through Matrigel were inhibited in a dose-dependent manner with total block of invasion at 250 and of migration at 1,000 µg/ml. These results suggest that NM has therapeutic potential in the treatment of breast cancer.
PDF
