Ascorbate supplementation inhibits growth and metastasis of B16FO melanoma and 4T1 breast cancer cells in vitamin C-deficient mice

Abstract

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.

Modulation of MMP-2 and −9 secretion by cytokines, inducers and inhibitors in human melanoma A-2058 cells

“…In addition, NM components N-acetyl cysteine and selenium have been shown to inhibit tumor cell MMP-9 and invasive activities and migration of endothelial cells through the ECM (23)(24)(25). Ascorbic acid has been documented to modulate cancer cell and tumor growth as well as to prevent metastasis (26)(27)(28)(29)(30)(31) and low levels of ascorbic acid are found in cancer patients (32)(33)(34). The inhibitory effects of NM in comparison to EGCG alone on MMP-9 and MMP-2 secretion in unstimulated and PMA-stimulated A-2058 cells suggest enhancing effectiveness of EGCG when applied in a mixture with other natural compounds of similar and complementary mechanisms.…”

Vitamin C in Disease Prevention and Cure: An Overview

“…Further recent experimental studies have also found that ascorbate supplementation hinders metastasis, tumour growth and inflammatory cytokine secretion as well as enhanced encapsulation of tumours in Gulo KO mice [149,150]. Reports have shown that intravenous injection increases vitamin C concentration more than 70 times in relation to oral administration and effectiveness of treatment is linked to vitamin C concentration [12,145].…”

Effect of a nutrient mixture on the localization of extracellular matrix proteins in HeLa human cervical cancer xenografts in female nude mice

“…N-acetyl cysteine and selenium have demonstrated the ability to inhibit the expression of MMP-9 and invasive activities of tumor cells, as well as the migration of endothelial cells through ECM (31)(32)(33). Ascorbic acid has demonstrated cytotoxic and antimetastatic actions on malignant cell lines (34)(35)(36)(37)(38)(39), and it has been observed that the levels of ascorbic acid in cancer patients are low (40,41). Low levels of arginine, a precursor of nitric oxide (NO), can limit the production of NO, which has been shown to predominantly act as an inducer of apoptosis (42).…”