Brain tumors are highly aggressive, characterized by the secretion of high levels of matrix metalloproteinase (MMP)-2 and MMP-9 that degrade the extracellular matrix and basement membrane, allowing ...cancer cells to spread to distal organs. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activity. We investigated the roles of these in the regulation of MMP-2 and MMP-9 in human glioblastoma T-98G cells. Human T-98G cells were grown in DME supplemented with 15% fetal bovine serum and antibiotics in 24-well tissue culture plates. At near confluence, cells were washed with phosphate-buffered saline and incubated in serum-free media with: phorbol 12-myristate 13-acetate (PMA) at 10, 25, 50 and 100 ng/ml; tumor necrosis factor (TNF)-α and interleukin (IL)-1β at 0.1, 1, 10 and 25 ng/ml; lipopolysaccharide (LPS) at 10, 25, 50 and 100 µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10, 25, 50 and 100 µM without and with PMA; a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract without and with PMA at 10, 50, 100, 500 and 1,000 µg/ml; actinomycin D and cyclohexamide at 2 and 4 µM; retinoic acid and dexamethasone at 50 µM. After 24 h the media were removed and analyzed for MMP-2 and MMP-9 by zymography and densitometry. Glioblastoma T-98G cells expressed only one band corresponding to MMP-2. PMA treatment showed increased MMP-2 and MMP-9 secretions up to 25 ng/ml and decreased levels of secretions at 50 and 100 ng/ml, with no significant overall effect. TNF-α induced an up and down effect on MMP-2 and a slight induction of MMP-9. IL-1β demonstrated a slight dose-dependent increase in T-98G secretion of MMP-2, but no induction of MMP-9. LPS showed dose-dependent decreased inactive MMP-2 secretion, increased active MMP-2 secretion and no effect on MMP-9. EGCG, Dox and NM, without and with PMA, downregulated the expression of MMP-2 and MMP-9 in a dose-dependent manner. Actinomycin D, cyclohexamide, retinoic acid and dexamethasone also had inhibitory effects on MMP-2. Our results showed that cytokines, mitogens and inhibitors modulated T-98G cell MMP-2 and MMP-9 expression, suggesting the clinical use of MMP inhibitors, particularly such potent and non-toxic ones as the nutrient mixture and its component EGCG in the management of glioblastoma cancers.
Degradation of the extracellular matrix (ECM) plays a critical role in the formation of tumors and metastasis and has been found to correlate with the aggressiveness of tumor growth and invasiveness ...of cancer. Ascorbic acid, which is known to be essential for the structural integrity of the intercellular matrix, is not produced by humans and must be obtained from the diet. Cancer patients have been shown to have very low reserves of ascorbic acid. Our main objective was to determine the effect of ascorbate supplementation on metastasis, tumor growth and tumor immunohistochemistry in mice unable to synthesize ascorbic acid gulonolactone oxidase (gulo) knockout (KO) when challenged with B16FO melanoma or 4T1 breast cancer cells. Gulo KO female mice 36-38 weeks of age were deprived of or maintained on ascorbate in food and water for 4 weeks prior to and 2 weeks post intraperitoneal (IP) injection of 5×105 B16FO murine melanoma cells or to injection of 5×105 4T1 breast cancer cells into the mammary pad of mice. Ascorbate-supplemented gulo KO mice injected with B16FO melanoma cells demonstrated significant reduction (by 71%, p=0.005) in tumor metastasis compared to gulo KO mice on the control diet. The mean tumor weight in ascorbate supplemented mice injected with 4T1 cells was reduced by 28% compared to tumor weight in scorbutic mice. Scorbutic tumors demonstrated large dark cores, associated with increased necrotic areas and breaches to the tumor surface, apoptosis and matrix metalloproteinase-9 (MMP-9), and weak, disorganized or missing collagen I tumor capsule. In contrast, the ascorbate-supplemented group tumors had smaller fainter colored cores and confined areas of necrosis/apoptosis with no breaches from the core to the outside of the tumor and a robust collagen I tumor capsule. In both studies, ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum inflammatory cytokine interleukin (IL)-6 (99% decrease, p=0.01 in the B16F0 study and 85% decrease, p=0.08 in the 4T1 study) compared to the levels in gulo KO mice deprived of ascorbate. In the B16FO study, ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum VEGF (98% decrease, p=0.019 than in the scorbutic gulo KO mice). As expected, mean serum ascorbate level in ascorbate-restricted mice was 2% (p<0.001) of the mean ascorbate levels in supplemented mice. In conclusion, ascorbate supplementation hinders metastasis, tumor growth and inflammatory cytokine secretion as well as enhanced encapsulation of tumors elicited by melanoma and breast cancer cell challenge in gulo KO mice.
Melanoma, an extremely aggressive cancer, causes the most skin cancer-related deaths, due to metastasis to other areas of the body, such as lymph nodes, lungs, liver, brain or bone. It is ...characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the extracellular matrix and basement membrane, allowing cancer cells to spread to distal organs. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activities. We investigated the roles of these in regulation of MMP-2 and -9 in human melanoma A-2058 cells. Human A-2058 cells were grown in DMEM supplemented with 15% FBS and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with PBS and incubated in serum-free media with phorbol 12-myristate 13-acetate (PMA) at 10, 25, 50 and 100 ng/ml; TNF-α and IL-1β at 0.1, 1, 10 and 25 ng/ml; LPS at 10, 25, 50 and 100 µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10, 25, 50 and 100 µM without and with PMA; a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract without and with PMA at 10, 50, 100, 500 and 1,000 µg/ml; actinomycin D and cyclohexamide at 2 and 4 µM; retinoic acid and dexamethasone at 50 µM. After 24 h the media were removed and analyzed for MMP-2 and MMP-9 by zymography and densitometry. Melanoma A-2058 demonstrated strong expression of MMP-2 and slight expression of MMP-9. PMA at 100 ng/ml showed no effect on MMP-2 secretion but potently upregulated MMP-9 secretion to 400% that of control. TNF-α showed no significant overall effect on expression of MMP-2 but potent dose-dependent increased MMP-9 secretion with 200% of control at 25 ng/ml. IL-1β showed no significant effect on MMP-2 or MMP-9 secretion by A-2058 cells, except at 25 ng/ml where MMP-2 level was reduced by ~40% and MMP-9 secretion ~50%. LPS treatment showed no significant effect on MMP-2 secretion and enhanced MMP-9 secretion up to 25 µg/ml followed by decreased level. EGCG, NM and doxycycline, without and with PMA, downregulated the expression of MMP-2 and MMP-9 in a dose-dependent manner. Actinomycin D, cyclohexamide and retinoic acid had inhibitory effects on MMP-2, while dexamethasone showed slight stimulatory effect on MMP-2 secretion. Our results showed that select cytokines, mitogens and inhibitors modulated A-2058 MMP-2 and MMP-9 expression. They suggest the clinical potential of MMP inhibitors, especially the non-toxic ones, such as the nutrient mixture and its component EGCG in management of melanoma.
Background: Tumor metastasis is a major cause for most cancer-related deaths. Melanoma is a serious cancer that metastasizes to other areas of the body, including the lungs, liver, brain, bones, or ...lymph nodes. Currently used cancer therapies are ineffective with a high degree of toxicity and patient mortality. Thus, any successful treatment for melanoma must target metastasis. Methods: We studied the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, green tea extract, quercetin, and others, on the inhibition of melanoma growth and metastasis after inoculation of B16FO melanoma cells into the left kidney of female nude mice. Female athymic mice (n = 10) 8 to 10 weeks of age, were inoculated superficially in the left kidney with 5 × 105 B16FO melanoma cells in 100 µL of media. The right kidney was left untreated. After inoculation, the mice were randomly divided into 2 groups. The control group (n = 5) was fed a regular rodent chow diet, and the test group was given the same diet supplemented with 0.5% NM. The animals in control and the test groups were sacrificed 2 weeks later. Each animal’s abdominal cavity was opened, and the kidneys, lungs, liver, and spleen were excised and examined for tumor growth and metastasis. Results: The kidneys in the control group weighed 25% to 30% more than those in the NM group due to colonization of B16FO melanoma cells. No metastasis to the liver or spleen was observed in either of the groups. However, severe lung metastasis was observed in the control group and mild to moderate metastasis was observed in the NM group. Conclusion: These results show that the NM is effective in mitigating the growth of tumors in the kidney and metastases to the lung.
Cancer progression is characterized by loss of extracellular matrix (ECM) integrity, which is a precondition for tumor growth and metastasis. In order to elucidate the precise mechanisms of ECM ...degradation in cancer we used a genetically modified mouse mimicking two distinct human metabolic features associated with carcinogenesis, the lack of endogenous vitamin C synthesis and the production of human Lp(a). Female Lp(a)+; Gulo(−/−) and control wild-type Balb/c mice without these two metabolic features were orthotopically inoculated with 4T1 breast cancer cells (5×105). The transgenic and control mice were divided into 4 different dietary groups in respect to dietary vitamin C intake: i) low ascorbate intake for 6 weeks; ii) high ascorbate intake for 6 weeks; iii) low ascorbate intake for 3 weeks followed by high ascorbate for 3 weeks; iv) high ascorbate intake for 3 weeks followed by low ascorbate for 3 weeks. After 6 weeks, all wild-type mice developed tumors. In contrast, Lp(a)+; Gulo(−/−) mice developed one third less primary tumors (low ascorbate diet) or no primary tumors at all (high ascorbate diet). Significantly, tumors from Lp(a)+; Gulo(−/−) mice immunostained positively for Lp(a) and their size was inversely proportional to Lp(a) serum levels. The results implicate that Lp(a) may play a role in controlling tumor growth and expansion. The most likely mechanism is the competitive inhibition of plasmin-induced ECM degradation due to the homology of Lp(a) components to plasminogen. The confirmation of this pathomechanism could lead to a universal therapeutic target for the prevention and treatment of cancer.
Cancers of the breast, cervix, uterus and ovary are the most prevalent cancers in women worldwide. 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 urokinase plasminogen activators (u-PA) and matrix metalloproteinases (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 human breast, cervix, uterine and ovarian cancer cell lines. Human breast (MDA-MB-231 and MCF-7), cervical (HeLa), uterine (SK-UT-1) and ovarian (SKOV3) cancer 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 breast and uterine 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 HeLa and SK-OV-3 cell lines. On gelatinase zymography, MDA-MB-231 and MCF-7 showed one band corresponding to MMP-9, HeLa showed two bands, an intense band corresponding to MMP-2 and a faint band corresponding to MMP-9, SK-UT-1 showed PMA-induced MMP-9, and SK-OV-3 showed a band corresponding to MMP-2. NM inhibited their expression in all cell lines. The activity of TIMPs was upregulated 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 female cancers.
Pediatric 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 activity of u-PA, MMPs and TIMPs in various human pediatric sarcomas. Human osteosarcoma MNNG-HOS, osteosarcoma U-2OS and rhabdomyosarcoma RD 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. All sarcoma cell lines studied expressed u-PA, which was inhibited by NM in a dose-dependent manner. On gelatinase zymography, osteosarcoma MNNG-HOS showed a band corresponding to MMP-2 and induction of MMP-9 with PMA (100 ng/ml) treatment. U-2OS osteosarcoma cells showed strong bands corresponding to inactive MMP-2 and MMP-9 and faint bands corresponding to active MMP-2 and MMP-9 dimer; PMA treatment enhanced MMP-9 and MMP-9 dimer activity. Rhabdomyosarcoma showed MMP-2 and faint MMP-9 bands; PMA treatment enhanced MMP-9 expression. 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 pediatric sarcomas.
Colorectal, pancreatic and hepatic carcinomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions, allowing cancer cells to spread to distal organs. These and other ...proteases, such as uPA, 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 uPA 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 activity of uPA, MMPs and TIMPs in colon HCT-116, pancreatic MIA PaCa-2 and hepatic carcinoma SK-Hep-1 cell lines. These cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 μg/ml. Analysis of uPA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. All three cancer cell lines expressed uPA, which was inhibited by NM in a dose-dependent manner. On gelatinase zymography, SK-Hep-1 showed bands corresponding to MMP-2 and MMP-9 with enhancement of MMP-9 with PMA (100 ng/ml) treatment. HCT-116 and MIA PaCa-2 showed strong bands corresponding to MMP-9 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 uPA and MMP-9 and a negative correlation between uPA/MMP-9 and TIMP-2. These findings suggest the therapeutic potential of NM in treatment of colon, pancreatic and hepatic carcinomas.
The prognosis of hepatocellular carcinoma (HCC) remains dismal despite any treatment. Matrix metalloproteinases (MMPs) have been researched for their role in tumor invasion and metastasis. Various ...cytokines, mitogens, growth factors, inducers, and inhibitors control MMP activities. In this article, we investigated the roles of these in the regulation of MMP-2, -9 secretions in HCC. Human HCC SK-Hep-1 was grown in appropriate media. At near confluence, the cells were washed with phosphate-buffered saline and incubated in serum-free media with PMA; TNF-α, IL-1β; lipopolysaccharide; epigallocatechin gallate (EGCG) and doxycycline (Dox) at various doses with and without PMA; a nutrient mixture (NM) containing lysine, proline, ascorbic acid, and EGCG with and without PMA at; and actinomycin D and cycloheximide at different doses. After 24 hours, the media were removed and analyzed. SK-Hep-1 expressed bands corresponding to MMP-2 and MMP-9. TNF-α showed an insignificant effect on MMP-2 at doses below 25 at which dose MMP-2 was virtually blocked and a moderate dose-dependent effect on MMP-9. Interleukin-1β demonstrated an insignificant effect on MMP-2 at doses below 25 at which dose MMP-2 was completely blocked and enhanced effects on MMP-9. Lipopolysaccharide showed dose-dependent inhibition of MMP-2 and MMP-9. EGCG, Dox, and NM, without and with PMA, downregulated the expression of MMP-2 and MMP-9. Actinomycin D and cycloheximide also had dose-dependent inhibitory effects on MMPs. Our results showed that cytokines, mitogens, and inhibitors modulated SK-Hep-1 MMP-2 and MMP-9 secretion, suggesting the clinical use of especially potent and nontoxic MMP inhibitor as the NM in management of HCC.
Brain tumors are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the extracellular matrix (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 urokinase plasminogen activators (uPA) 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 uPA, MMPs and TIMPs in various human gliomas. Human glioblastoma (LN-18, T-98G and A-172) cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 μg/ml. Analysis of uPA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Glioblastoma cell lines LN-18 and T-98G expressed uPA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to uPA were detected for the A-172 cell line. On gelatinase zymography, all three cell lines showed bands corresponding to MMP-2 and LN-18 and T-98G showed PMA (100 ng/ml)-induced MMP-9. NM inhibited their expression in a dose-dependent manner. Activity of TIMP-2 was upregulated by NM in all glioma cell lines in a dose-dependent manner. Analysis revealed a positive correlation between uPA and MMP-2 and a negative correlation between uPA/MMPs and TIMP-2. These findings suggest the therapeutic potential of NM in the treatment of gliomas.
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