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

Degradation of the extracellular matrix (ECM) plays a critical role in theformation of tumors and metastasis and has been found to correlate with the aggressivenessof tumor growth and invasiveness of cancer. Ascorbic acid, which is known to beessential for the structural integrity of the intercellular matrix, is not producedby humans and must be obtained from the diet. Cancer patients have been shownto have very low reserves of ascorbic acid. Our main objective was to determinethe effect of ascorbate supplementation on metastasis, tumor growth and tumorimmunohistochemistry in mice unable to synthesize ascorbic acid [gulonolactoneoxidase (gulo) knockout (KO)] when challenged with B16FO melanoma or 4T1 breastcancer cells. Gulo KO female mice 36-38 weeks of age were deprived of or maintainedon ascorbate in food and water for 4 weeks prior to and 2 weeks post intraperitoneal(IP) injection of 5x105 B16FO murine melanoma cells or to injection of 5x105 4T1breast cancer cells into the mammary pad of mice. Ascorbate-supplemented gulo KOmice injected with B16FO melanoma cells demonstrated significant reduction (by71%, 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 wasreduced by 28% compared to tumor weight in scorbutic mice. Scorbutic tumors demonstratedlarge dark cores, associated with increased necrotic areas and breaches to thetumor surface, apoptosis and matrix metalloproteinase-9 (MMP-9), and weak, disorganizedor missing collagen I tumor capsule. In contrast, the ascorbate-supplemented grouptumors had smaller fainter colored cores and confined areas of necrosis/apoptosiswith no breaches from the core to the outside of the tumor and a robust collagen Itumor capsule. In both studies, ascorbate supplementation of gulo KO mice resultedin 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) comparedto the levels in gulo KO mice deprived of ascorbate. In the B16FO study, ascorbatesupplementation 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 serumascorbate level in ascorbate-restricted mice was 2% (p<0.001) of the mean ascorbatelevels in supplemented mice. In conclusion, ascorbate supplementation hindersmetastasis, tumor growth and inflammatory cytokine secretion as well as enhancedencapsulation of tumors elicited by melanoma and breast cancer cell challengein gulo KO mice.