Elsevier

Life Sciences

Volume 75, Issue 8, 9 July 2004, Pages 955-967
Life Sciences

Inhibition of the development of metastases by dietary vitamin C:K3 combination

https://doi.org/10.1016/j.lfs.2004.02.011 Get rights and content

Abstract

The tumor growth-inhibiting and chemo-potentiating effects of vitamin C and K3combinations have been demonstrated both in vitro and in vivo. The purpose of this study was to investigate the influence of orally administered vitamin C and K3 on the metastasis of mouse liver tumor (T.L.T.) cells implanted in C3H mice. Adult male C3H mice were given water containing vitamin C and K3 (15 g/0.15 g dissolved in 1000 ml) beginning 2 weeks before tumor transplantation until the end of the experiment. T.L.T. cells (106) were implanted intramuscularly in the right thigh of mice. All mice were sacrificed 42 days after tumor transplantation. Primary tumor, lungs, lymph nodes and other organs or tissues suspected of harboring metastases were macroscopically examined. Samples of primary tumors, their local lymph nodes, lungs and main organs such as liver, kidneys, spleen were taken for histological examination. Forty-two percent of control mice exhibited lung metastases and 27% possessed metastases in local lymph nodes whereas 24% of vitamin-treated mice exhibited lung metastases and 10% possessed local lymph nodes metastases. The total number of lung metastases was 19 in control group and 10 in vitamin C and K3-treated mice. Histopathological examination of the metastatic tumors from the vitamin-treated mice revealed the presence of many tumor cells undergoing autoschizic cell death. These results demonstrate that oral vitamin C and K3 significantly inhibited the metastases of T.L.T. tumors in C3H mice. At least a portion of this inhibition was due to tumor cell death by autoschizis.

Introduction

The influence of dietary components on tumor growth and development has recently become a subject of major interest Williams and Dickerson, 1990, Roberfroid, 1991, Milner, 1994. Amongst different alimentary factors, vitamin C and vitamin K3 were investigated as possible antitumor agents Park et al., 1980, Prasad et al., 1981, Gold, 1986. These vitamins were of particular interest because vitamin C (ascorbic acid or sodium ascorbate) was shown to exclusively reactivate acid DNase (DNase II) in malignant tumor cells, while vitamin K3 (2-methyl-1-4-naphthoquinone) selectively reactivated alkaline DNase (DNase) Taper, 1980, Taper et al., 2001.

These observations were of great interest because previously published studies demonstrated that the activity of alkaline and acid DNases was inhibited in non-necrotic cells of malignant tumors in human and experimental animals as well as during early stages of experimental carcinogenesis Taper, 1967, Taper et al., 1971a, Taper et al., 1971b, Fort et al., 1974, Taper and Bannasch, 1976. Furthermore, reactivation of alkaline and acid DNase-induced tumor cell death and tumor regression Taper, 1980, Taper et al., 1981. In fact, the tumor growth-inhibiting and chemotherapy-potentiating effects of vitamin C and K3combinations were evaluated using a variety of human tumor cell lines Noto et al., 1989, De Loecker et al., 1993. These in vitro studies were extended to a battery of human urologic tumor cell lines, including DU145, an androgen-independent prostate carcinoma cell line Gilloteaux et al., 1995, Gilloteaux et al., 1999, Venugopal et al., 1996a, Venugopal et al., 1996b, Jamison et al., 1996, Jamison et al., 1997, Jamison et al., 2001. In these studies, autoschizis, a new type of cell death which differs from necrosis and apoptosis, was described Gilloteaux et al., 1998, Gilloteaux et al., 1999, Gilloteaux et al., 2001a, Gilloteaux et al., 2001b, Jamison et al., 2001.

In vivo administration of the vitamin C and K3 combination to ascites tumor-bearing mice (at a single, intraperitoneal dose of vitamin C = 1g/Kg body weight and vitamin K3 = 0.01 g/Kg) synergistically increased the life span of mice by 45% when compared with sham treated control mice. Individual administration of vitamin C and vitamin K3increased life span by 14% and 1.07%, respectively (Taper et al., 1987). Combined vitamin C and K3 administration also potentiated the chemotherapeutic effects of six different cytotoxic drugs commonly utilized in the classical protocols of human cancer treatment (Taper et al., 1987). The same vitamin C and K3 combination also sensitized a mouse tumor that was resistant to vincristine and potentiated the therapeutic effects of radiotherapy Taper and Roberfroid, 1992, Taper et al., 1996. More recently, vitamin C administration with a benzoquinone was shown to inhibit the metastasis of several colon cancer cell lines that had been implanted into immunocompetent mice (Hidvégi et al., 1998). The structural similarity of vitamin K3 (a naphthoquinone) to the benzoquinone employed in these studies suggested that the vitamin C and K3 combination may decrease tumor metastasis as well as inhibiting the growth of the primary tumor.

Metastases are one of the greatest problems in cancer patients. They appear frequently and are the primary cause of mortality in cancer patients (Fidler, 1999). Different mechanisms are involved in the so-called metastatic cascade, including angiogenesis, cellular adhesion, local proteolysis and tumor cell migration Kohn, 1993, Fidler, 1999. Development of chemotherapeutic agents which target and intervene in one or more processes in the metastatic cascade should lead to a favorable outcome for a large number of cancer patients.

The current study evaluates the ability of orally administered vitamin C and K3 to inhibit the development of metastases of mouse liver tumor (TLT) cells that have been implanted into the thigh of C3H mice.

The TLT cell line is a primary liver tumor of spontaneous origin that was first observed in a 2 month old female Swiss-Webster mouse. The TLT tumor was derived from in vivo passage in mice and characterized by Taper and his colleagues at Sloan Kettering Institute for Cancer Research (Taper et al., 1966). Tumor growth is rapid, invasive and not strain-specific. TLT cells were employed because these tumor cells provide an excellent model for metastasis. Unlike many putative metastatic models in which tumor cells are injected into the tail vein of immunosuppressed rodents and “metastasize” to the lungs, a solid primary tumor is formed in the TLT model. Subsequently, cells from the primary tumor recapitulate all the stages of metastasis.

Section snippets

Animals and diets

Young adult male C3H mice weighing 25–30 g at the beginning of experiments were purchased from IFFA Credo, Domaine des Oncins, France and were fed basal diet for experimental animals AO4, supplied by U.A.R., Villemoison-sur-Orge, France. Vitamin C (sodium ascorbate) and vitamin K3 (2-methyl-1,4-naphthoquinone) were purchased from Sigma, St. Louis, MO, USA. Control mice received water ad libitum. The mice in the experimental group received vitamin C and K3 (15 g/0.15 g) dissolved in 1000 ml of

Results

At early stages after tumor transplantation, vitamin C and K3 treatment produced a distinct inhibition of solid and ascitic TLT tumor growth without producing any distinct difference in morphology of treated and control TLT tumors Taper et al., 1987, Taper and Roberfroid, 1992, Taper et al., 1996. However, the progression of intramuscularly transplanted T.L.T. tumors in C3H mice rapidly became lethal and obliged the sacrifice of all mice 42 days after tumor transplantation. This mortality was

Discussion

The results of the current study demonstrated that oral administration of vitamin C and K3 produced a distinct inhibitory effect on the development of metastases in C3H mice bearing an intramuscularly transplanted mouse liver tumor. Detailed microscopic examination of the main organs as well as the lungs and local lymph nodes (other tissues known to harbor metastases from this tumor) revealed a distinct inhibitory effect of oral vitamin treatment on the development of metastases. As was the

Acknowledgements

The authors thank Dr. J. Cumps for statistical analysis, Mrs. R.M. Dujardin for very competent technical assistance and Mrs. E. Ferdinand for careful typing of the manuscripts.

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