By Carrie Decker, ND
Founder and Medical Director, Blessed Thistle, Madison, WI
Dr. Decker reports no financial relationships relevant to this field of study.
SUMMARY POINTS
- A series of experiments using human-sourced melanoma cells were developed to investigate the mechanisms promoting metastasis in vivo in mice.
- Highly efficient melanoma cell lines had low rates of metastasis when injected intravenously (1:235) or directly into visceral organs (variable by organ, 1:173 for intrasplenic injection), while subcutaneous injections led to formation of local tumors at a much higher rate (1:8).
- Mice with subcutaneous melanoma tumors that were treated with daily injections of a known antioxidant, N-acetyl-cysteine (NAC), had an increased metastatic burden.
- Pre-treatment of melanoma cells with NAC prior to intravenous injection increased tumor formation tenfold.
SYNOPSIS: A series of experiments utilizing human melanoma cells found that oxidative stress was higher in circulating melanoma cells and distant metastasis than the original subcutaneous tumors, suggesting a higher oxidative stress burden subsequent to leaving the original tumor environment. Treatment of animals or tumor cell lines with antioxidants led to increased metastasic burden.
SOURCE: Piskounova E, et al. Oxidative stress inhibits distant metastasis by human melanoma cells. Nature 2015;527:186-191.
Human cells from highly efficient (UT10, M481, M405, and M514) and inefficient (M597, M528, M610, and M498) metastasizing melanomas were used for a series of experiments investigating the mechanisms promoting metastasis in animal studies. In the first set of experiments, cells from the highly efficient and inefficient cell lines were injected into mice subcutaneously, intravenously, or directly into visceral organs. Cell lines with known inefficient metastatic potential were found to be associated with much lower levels of distant metastasis with injection subcutaneously, intravenously, and even direct injection into visceral organs, reinforcing clinical observations. However, the inefficient and efficiently metastasizing cell lines did not exhibit a difference in solid tumor formation or growth rate with local subcutaneous injection, suggesting the differences in metastatic potential is due to cellular differences that occur outside the original environment.
In the second set of experiments, the potential reversibility of cellular changes associated with higher rates of metastasis was investigated. Cells from a single melanoma tumor line that had formed subcutaneous tumors, were present in circulation, and had formed liver metastasis were given to recipient mice by subcutaneous, intravenous, or intrasplenic injection in a full factorial experiment. The primary finding of this set of experiments was that the melanoma tumor cells were most likely to form new tumors in the environment from which they were sourced (i.e., cells from a metastatic nodule were most likely to form a tumor when injected in a distant organ, and subcutaneous tumor cells were most likely to form a new subcutaneous tumor).
With a follow-up experiment to investigate if changes were reversible, melanoma cells from subcutaneous tumors, the blood, or liver metastasis were transplanted subcutaneously in recipient mice and allowed to form subcutaneous tumors. Cells from these subcutaneous tumors of differing original sources were then transplanted to a new set of recipient mice by subcutaneous, intravenous, and intrasplenic injection. Cells from all original cell lines formed subcutaneous tumors with high efficiency while metastatic tumors were formed at a very low efficiency. Thus, the changes that the cells experienced leading to their metastatic potential are likely reversible.
Finally, the levels of oxidative stress in melanoma cells and the effect of antioxidant therapies were investigated. In subcutaneous tumors, a significantly higher glutathione to oxidized glutathione ratio was found compared to the circulating melanoma cells (CMCs, P < 0.05) and those from metastatic nodules (P < 0.0005). Additionally, higher levels of cytoplasmic reactive oxygen species (ROS) were found in CMCs and distant metastatic cells, and higher mitochondrial ROS levels were found in metastatic cells (P < 0.00005), suggesting a higher oxidative stress burden in distant locations. A decreased mitochondrial mass was found in the CMCs and metastatic cells. As mitochondrial respiration is a main source of ROS, it suggests this is a mechanism by which the melanoma cells attempt to reduce ROS burden.
To investigate the effect of antioxidants, after subcutaneous transplant of previously metastatic melanoma cell lines, N-acetyl-cysteine (NAC) was given by subcutaneous daily injections at a level of 200 mg/kg/day. NAC treatment did not significantly affect the growth of subcutaneous tumors, but it did increase levels of CMCs with some cell lines as well as metastasis burden with all cell lines tested (P < 0.05 to 0.0005, depending on cell line). Pretreatment of efficiently metastasizing melanoma cells with NAC prior to intravenous injection was associated with a tenfold increase in tumor formation (P < 0.0001). The combination of these factors suggests oxidative stress plays a significant role in the inhibition of melanoma metastasis.
In addition to these experiments, further investigations of the mechanisms that potentially contribute to glutathione regeneration were performed. Cellular adaptations that may support glutathione levels were found in efficiently metastasizing cell lines, including increased NADPH (P < 0.05) and higher levels of the NADPH regenerating enzyme ALDH1L2. Other changes suggested increased utilization of the folate pathway for NADPH regeneration. MTHFD1 also is a NADPH regenerating enzyme in the folate pathway, and gene knockdown studies of ALDH1L2 and MTHFD1 found that both of these enzymes were significant in promoting melanoma metastasis. Finally, treatment with low-dose methotrexate, an inhibitor of dihydrofolate reductase, was found to significantly reduce the frequency of CMCs and metastatic burden, while it did not have a significant effect on the growth of subcutaneous tumors.
COMMENTARY
Simply summarizing the rather complex series of experiments, what this study suggests is that a higher oxidative stress burden is, in part, what limits melanoma metastasis, and cells that more efficiently metastasize experience metabolic changes that support their own antioxidant production. Antioxidant treatment of animals and cell lines also promoted metastasis. Although this study investigates only melanoma cell lines, the issues it raises concerning oxidative stress and metastasis are pertinent, as antioxidant therapies are often discussed as part of the treatment and prevention of cancer. Antioxidants are often used to reduce side effects of radiation or chemotherapy by protecting healthy tissues from damage induced by oxidative stress. Administration of antioxidants during radiation and chemotherapy is a controversial issue, as the success of treatments such as these are often based in part on the damage induced by oxidative stress to cancerous cells.1 The findings of this study lead to additional concerns with the use of antioxidants in the setting of cancer treatment or prevention, as treatment of both the animal and cell with antioxidants increased metastasis significantly.
As oxidative stress is a mechanism by which oncogenic mutations in cellular DNA may occur,2,3 antioxidants are obviously not something to be avoided at all costs for the purpose of cancer prevention. Oxidative stress within cells and related ROS may even act as secondary messengers, inducing and maintaining the oncogenic phenotype of cancer cells.4 The oxidative stress associated with radiation therapies for cancer are associated with secondary carcinogenesis.5 It has been estimated that the cumulative incidence of secondary malignancies could be as high as 20% in patients treated by radiotherapy.6 In the past, the incidence of radiation-induced secondary malignancies had been underestimated because most patients had a short life expectancy after treatment.
Other studies investigating the use of antioxidant therapies have been neutral or showed benefits with their use as an adjunctive treatment for cancers (including those with metastasis). Intravenous (IV) treatment with alpha-lipoic acid has been used in the setting of pancreatic cancer with metastasis,7 while IV ascorbic acid has been studied for use in a variety of cancer treatment settings.8.9.10 Antioxidant therapies have been studied in stage 3 and 4 cancer patients with various malignancies and were shown to reduce reactive oxygen species and serum levels of inflammatory cytokines while increasing glutathione peroxidase activity; however, in this study, the authors did not investigate the effects on outcomes.11 Lipoic acid also has been studied in clinical trials for the prevention of chemotherapy-induced peripheral neuropathy without significant adverse effects or worsening the outcomes of chemotherapy,12 while NAC has been used for prevention of cisplatin-induced ototoxicity and oxaliplatin-induced neuropathy.13,14
A review pertaining to the use of vitamins, particularly vitamins A, D, E, K, and C, in the setting of melanoma also was recently published.15 Each of these can have some action as an antioxidant, although vitamin E and C are the ones most often thought of as such. Multiple studies, both in vitro and in vivo, support the use of these nutrients as protective agents or useful for chemoprevention and treatment for melanoma, although the mechanisms by which they may be beneficial vary. However, vitamin C in particular has been shown to support the cellular levels of glutathione, which was shown to have a detrimental effect in the current study.16 Other compounds with known antioxidant potential, such as green tea catechins,17 resveratrol,18 and curcumin,19 have been studied extensively in the setting of cancer (including melanoma) and have evidence supporting their use, despite also functioning as antioxidants. Finally, the dietary intervention of the Gerson diet, an organic plant-based diet with frequent consumption of raw vegetable/fruit juices, was assessed in a retrospective review of the 5-year survival rates of melanoma patients, and survival rates were considerably higher than what is typical.20
This is not the first setting in which antioxidants have been questioned as a therapy. Although many studies show isolated parameter and clinical improvements with antioxidant therapies, other studies associate antioxidants with higher mortality as well.21 Oxidative stress itself plays an important role in instigating cellular repair and antioxidant transcription.22 In a recent publication it was shown that cancer risk is potentially 70-90% influenced by extrinsic rather than intrinsic factors.23 Thus, the potential contribution of oxidative stress associated with carcinogens, infection, and lifestyle choices is also likely high.24 Although cancer patients typically have higher oxidative stress and lower antioxidant activity, oxidative stress markers have not been associated with survival of terminally ill cancer patients.25
As more and more knowledge is gained in the field of oncology through studies such as these to understand the differences between various cancerous cell lines, we can continue to anticipate further specialization of cancer treatment strategies, including nutritional support. Given the findings of this study, individuals with melanoma, especially those with known metastasis, may want to avoid supplemental antioxidants, particularly NAC, until further research pertaining to the findings of this study can be done.
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- Russo I, et al. Vitamins and melanoma. Cancers (Basel) 2015;7:1371-1387.
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- Singh T, et al. Green tea catechins reduce invasive potential of human melanoma cells by targeting COX-2, PGE2 receptors and epithelial-to-mesenchymal transition. PLoS One 2011;6:e25224.
- Osmond GW, et al. Enhancing melanoma treatment with resveratrol. J Surg Res 2012;172:109-115.
- Loch-Neckel G, et al. Orally administered chitosan-coated polycaprolactone nanoparticles containing curcumin attenuate metastatic melanoma in the lungs. J Pharm Sci 2015;104:3524-3534.
- Hildenbrand GL, et al. Five-year survival rates of melanoma patients treated by diet therapy after the manner of Gerson: A retrospective review. Altern Ther Health Med 1995;1:29-37.
- Bjelakovic G, et al. Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases. Cochrane Database Syst Rev 2012;14:CD007176.
- Nguyen T, et al. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J Biol Chem 2009;284:13291-13295.
- Wu S, et al. Substantial contribution of extrinsic risk factors to cancer development. Nature 2015 Dec 16. [Epub ahead of print].
- Reuter S, et al. Oxidative stress, inflammation, and cancer: How are they linked? Free Radic Biol Med 2010;49:1603-1616.
- Yeom CH, et al. Oxidative stress level is not associated with survival in terminally ill cancer patients: A preliminary study. BMC Palliat Care 2014;13:14.
A series of experiments utilizing human melanoma cells found that oxidative stress was higher in circulating melanoma cells and distant metastasis than the original subcutaneous tumors, suggesting a higher oxidative stress burden subsequent to leaving the original tumor environment. Treatment of animals or tumor cell lines with antioxidants led to increased metastasic burden.
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