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.

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The highly aggressive pediatric sarcomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and MMP-9, which play crucial roles in tumor invasion and metastasis by degradation of the extracellular membrane leading to cancer cell spread to distal organs. We examined the effects of cytokines, mitogens, inducers and inhibitors on MMP-2 and -9 expression in osteosarcoma (U2OS) and rhabdomyosarcoma (RD). The selected compounds included natural cytokines and growth factors, as well as chemical compounds applied in therapy of sarcoma and natural compounds that have demonstrated anticancer therapeutic potential. These cell lines were cultured in their respective media to near confluence and the cells were washed with PBS and incubated in serum-free medium with various concentrations of several cytokines, mitogens and inhibitors. After 24 h the media were removed and analyzed for MMP-2 and -9 by gelatinase zymography and quantitated by densitometry. Osteosarcoma and rhabdomyosarcoma showed bands corresponding to MMP-2 and -9 with dose-dependent enhancement of MMP-9 with phorbol 12-myristate 13-acetate (PMA) treatment. Tumor necrosis factor-α, interleukin-1β and LPS enhanced osteosarcoma U2OS MMP-9 secretion but had no effect on MMP-2 secretion. Tumor necrosis factor-α stimulated rhabdomyosarcoma MMP-2 expression, but had no effect on MMP-9 secretion. Doxycycline, epigallocatechin gallate, nutrient mixture (NM), actinomycin-D, cyclohex-amide, retinoic acid and dexamethasone inhibited MMP-2 and -9 in U2OS osteosarcoma cells. PMA-treated RD cells showed dose-response inhibition of MMP-9 by doxycycline and epigallocatechin gallate and both MMPs by NM. Dexamethasone and actinomycin-D showed inhibition of MMP-2 secretion of RD cells. Our results show that cytokines, mitogens and inducers show variable upregulation of U2OS osteosarcoma and RD rhabdomyosarcoma MMP-2 and -9 secretion, and inhibitors demonstrate downregulation under stimulatory conditions, suggesting the application of these agents for the development of effective therapies in pediatric sarcomas.

Current treatment of fibrosarcoma, an aggressive cancer of the connective tissue, is generally associated with poor prognosis. Matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), and constituents of the extracellular matrix (ECM), such as fibronectin, play a critical role in angiogenesis and underlie neoplastic invasion and metastasis. This and anticancer properties of lysine, proline, arginine, ascorbic acid, and green tea extract (NM) prompted us to investigate the effect of these nutrients in vitro on human fibrosarcoma cells HT-1080 by measuring cell proliferation, modulation of MMP-2 and MMP-9, and invasive potential. In vivo, we studied the growth of human fibrosarcoma HT-1080 cells in athymic nude mice and the expression of MMPs and VEGF. Cell proliferation was evaluated by MTT assay, MMP expression by gelatinase zymography, and invasion through Matrigel and migration by scratch assay. Tumors were excised, weighed, and processed for histology in both the control and nutrient-supplemented groups. Results showed NM inhibited the growth and reduced the size of tumors in nude mice; decreased MMP-9 and VEGF secretion was found in the supplemented group tissues. NM inhibited invasion through Matrigel and migration with total inhibition at 1,000 microg/mL. These results offer promise in the therapeutic use of the nutrient mixture of lysine, proline, arginine, ascorbic acid, and green tea extract tested in the treatment of fibrosarcoma.

Current treatments are generally ineffective once breast cancer has metastasized; median survival is reduced to 2-3 yr. Previous research studies demonstrating potent synergistic antitumor activity of lysine, proline, ascorbic acid, and epigallocatechin gallate prompted us to investigate the in vivo inhibitory effect of a nutrient mixture containing lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate (NM) on the growth of human cancer xenografts in female athymic nude mice. Five to six week old female mice were inoculated with 3x106 breast cancer cells MDA-MB-231. After injection, the mice were randomly divided into two groups A and B; group A was fed a regular diet and group B with the regular diet supplemented with 0.5% of the nutrient mixture (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 on estrogen-receptor positive (ER+) MCF-7 and estrogen-receptor negative (ER-) MDA-MB-231 breast cancer cell lines by measuring: cell proliferation by MTT assay, expression of MMPs by gelatinase zymography, invasion through Matrigel, and VEGF by ELISA. MCF-7 cells were also treated with estradiol to study enhanced invasion and expression of MMPs and VEGF. Results showed that NM inhibited the growth and reduced the size of tumors in female nude mice by 27%. Furthermore, histological evaluation revealed increased mitotic index, MMP-9 and VEGF secretion, and PAS material (mucin) in the control group tissues. In vitro studies showed NM inhibited MDA-MB-231 cell growth by 34% at 500 microg/mL and MCF-7 cell growth by 18% at 1000 microg/mL. Invasion of MDA-MB-231 through Matrigel was inhibited by 50%, 60%, and 95% by 10, 50, and 100 microg/mL of NM, respectively. The results of this study demonstrated that the nutrient mixture tested significantly suppressed tumor growth of breast cancer cells in female athymic nude mice and significantly inhibited MMP expression, angiogenesis, and invasion in breast cancer cells, in vitro, offering promise for therapeutic use in the treatment of breast cancer.