Nutrition Research, Vol. 20, No. 11, pp. 1557-1567, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA.  All rights reserved. 0271-5317/00/$---see front matter
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Effects Of The Coral Calcium As An Inhibitory Substance Against Colon Cancer And Its Metastasis In The Lungs

Results & Discussion One of the factors contributing to the inhibition of metastases formation of cancer cells is an immune system; i.e., cellular immunity (4, 5, 9 and 10) consisting of NK cells, T-cells, B-cells, macrophages, etc. In the present study, since the Coral Calcium was assumed to be involved in the change of pH in the extracellular fluid and cellular immunity such as NK cells and macrophages, the prophylactic effects of Coral Calcium against the onset of cancer were investigated in culture media using Colon 26 cancer cells as well as the formation of metastasis of the cancer cells in the lungs. Initially, the mouse Colon 26 cancer cells were cultured in vitro using various media adjusted to designated pH values with Coral Calcium, 1N NaOH and 1N HCl, for 120 hrs. The growth rate of the Colon 26 cancer cells in each group was investigated. The growth rates of the Colon 26 cancer cells in various groups were compared to that of the control group with a pH 7.2, which is the pH value of blood in healthy human supjects. As shown in Fig. 2, the growth rates ranged from 127.9% at pH 7.0, 143.6% at pH 6.5, and 142.8% at pH 6.0, with a pH of 6.5 having the maximum growth rate. In this study, the proliferation of cancer cells was observed at pH 6.5, but was rather slightly (p<0.01) inhibited at pH 6.0. (the experiment could not be performed below pH 6.0 since the cell membrane of cancer cell was destroyed.) This study suggests that pH 6.5 is the optimum pH for which the cancer cells are likely to proliferate. On the other hand, the growth rate in the weak alkaline group (pH 7.5) was 68.2%. thus resulting in the prevention of growth of cancer cells. In fact, the cancer cells did not proliferate under the same weak alkaline conditions as those found in the small intestine, thus supporting the fact that the onset of cancer is not observed in the small intestine, as based on a survey disclosed by the Ministry of Health and Welfare in Japan (1). When the morphologic (pathological) changes of the cancer cells were observed using a microscope at 120 hours after the cultivation, it was found that the cancer cells with a significant growth rate at pH 6.5 continuously proliferate (Photo I), while extending dendritic projections stereoscopically to the surrounding organs. In the group with pH 7.5, the cancer cells were individually scattered, along with inhibition of growth. Moreover, about half of the cancer cells enlarged to about 1.5-fold in size, with intracellular granules or vacuoles, or blackening (apoptosis) of cancer cells (Photo 2). These results have suggested that the weak alkaline pH prepared plays a significant role in inhibiting the proliferation of the cancer cells. Next, the effect of Coral Calcium concentration against Colon 26 cancer cells was subsequently investigated using calcium carbonate as a control. To each of the various pH solutions (containing 500mg of Coral Calcium per dL, as a basal amount), 5, 10 and 15 mg of Coral Calcium per dL were respectively added. Similarly, to each of the various pH solutions (containing 500 mg of Coral Calcium per dL), 5, 10 and 15 mg of calcium carbonate per dL were respectively added. The growth rates of Colon 26 cancer cells were measured in these solutions. As a result, the growth of the cancer cells tended (p<0.05) to be inhibited in the pH 7.5 group in which 15 mg of Coral Calcium per dL was added, compared to the other groups. Considerable progress has recently been made in cancer research. Some effective nutrients or chemicals with prophylactic effects on the onset of cancer have been found from dietary sources (11, 12, 13 and 14). From the perspective that cellular immunity induced by calcium relates to the inhibition of proliferation of the cancer cells, the inhibitory effects ofCoral Calcium against the formation of their metastases in the lungs were investigated, using the mouse Colon 26 cancer cells (in vivo). Prior to the start of this investigation, the following three in vivo studies were carried out as control studies. First, in the BALB/C mice (n=5) at the age of 7 weeks (with no treatment: i.e., no dosing of Coral Calcium and no inoculation of the cancer cells), the activity of NK cells and the number of macrophages were observed. The activity of NK cells was 11.9% on Day 7 and 12.3% on Day 14, whereas the number of macrophages was 3.5% on Day 7 and 2.4 on Day 14. The reason why the activity of NK cells was high and the number of macrophages was low was considered to be due to the lack of burden such as the administration of Coral Calcium or the inoculation of cancer cells. Second, the BALB/C mice (n=5) at the age of 7 weeks were intravenously inoculated only with Colon 26 cancer cells (1 x 105 cells for each mouse) for 14 days. Then the activity of NK cells and the number of macrophages were observed. The activity of NK cells was 5.9% on Day 7 and 4.5% on Day 14, whereas the number of macrophages was 5.8% on Day 7 and 4.3% on Day 14. These results showed a similarity to those of Group B. On histopathological findings, marked metastatic lesions in the lungs were observed along with some inflammatory cellular inflammation and some increases in neutrophile and monocyte counts. The level of serum albumin was measured and was found to be about 2.8 mg/dL, which is within normal range. (Since there were no changes in the level of serum albumin in this study and an amount of the blood was not sufficiently collected, we have not observed serum albumin as a parameter in the other studies.) Third, the effect of Coral Calcium, consisting of calcium and various trace elements, was investigated in comparison with calcium carbonate used as a control. The effects of Coral Calcium and calcium carbonate against the Colon 26 cancer cells were compared using BALB/C mice at the age of 7 weeks. Coral Calcium was orally administered at 100 mg/kg, 300 µL/body to each of 5 mice once a day for 14 consecutive days, whereas calcium carbonate was also orally administered at 100 mg/kg, 300 µL/body to each of 5 mice once a day for 14 consecutive days. On Day 14 after the administration, Colon 26 cancer cells (1 x 105 cells for each mouse) were intravenously inoculated to these two groups. Thereafter, on Day 7 after the inoculation, the activity of NK cells and the number of macrophages were observed along with an observation of the metastasis of cancer in the lungs. As a result, the activity of the NK cells and the number of macrophages in the group to which Coral Calcium was administered were greater (P<0.05) than those in the group to which calcium carbonate was administered. Finally, based on the results of the above three studies, the effects of Coral Calcium concentrations were investigated. Group A to which Coral Calcium was administered at 100 mg/kg, 300 µL/body, was compared to group S to which Coral Calcium was administered an 5 mg/kg, 300 µL/body. On Day 7 after the inoculation of cancer cells, the activity of NK cells in group A was about 1.8 times as much as that of NK cells in group B, whereas the number of macrophages in group A similarly increased (Fig. 3). On Day 14 after the inoculation, the number of metastatic lesions (nodules) of cancer cells in the lungs was 19.3 in group A and 65.5 in group B; i.e.. the incidence of the metastases in group A was about 1/3 less than that of group B (Fig. 4).  Interestingly, in group A, a remarkable increase in the number of monocytes was histopathologically observed. On histopathological findings, the proliferation of cancer cells was occasionally found in group B (Photo 3); i.e., the lesions of cancer cells in the pulmonary circumvascular and alveolar portions formed and infiltrated into the surrounding alveolus. On the other hand, although only a small number of cancer cells were observed in the pulmonary circumvascular and alveolar portions in group A, no metastatic lesions were observed in the lungs (Photo 4). From the above, in vitro study results, the weak alkaline pH was found to inhibit the proliferation of Colon 26 cancer cells significantly, thus supporting the fact that the onset of cancer is not observed in the small intestine. In the subsequent study (in vitro), the Colon 26 cancer cells tended to be inhibited more markedly by Coral Calcium than by calcium carbonate (as a control). In vivo studies, the activity of the NK cells and the number of macrophages in the group to which Coral Calcium was administered were greater than those in the group to which calcium carbonate was administered. Furthermore, the greater the dose of Coral Calcium, the greater the activity of NK cells and the number of macrophages increased. The metastasis of the cancer cells in the lungs was significantly inhibited at a higher dose of Coral Calcium. So far, some investigators (15, 16) have reported that calcium induces the activation of macrophages. From the present study results, it was assumed that calcium, as a major component in Coral Calcium, could possibly activate the NK cells and increase the number of macrophages, resulting in an inhibitory effect on 26 cancer cells (i.e., offensive to the cancer cells). The remarkable increase in the number of monocytes was histopathologically observed in group A (in vivo). As such, the monocytes are assumed to have activated the NK cells and increased the number of macrophages indirectly, resulting in the inhibition of the metastasis of cancer in the lungs. More interestingly, since there was a tendency for the cancer cells to be more inhibited by Coral Calcium than by calcium carbonate, it is considered than some trace elements contained in Coral Calcium could play a role in the inhibition of the cancer cells. The trace elements are known to have a wide variety of activities, as reported in 'Hand Book of Nutritionally Essential Mineral Elements (17).' Further investigation would be needed on the effects of the trace elements on the inhibition of cancer, as an important theme in terms of the science of nutrition. Judging from histological findings and nodulation of the lungs and hematological findings, the cancer cells were assumed to be attacked by NK cells and macrophages in the blood after their inoculation and before arrival to the lungs (target organs), so that most of the cancer cells did not result in the formation of metastases in the lungs. Thus, it has been suggested that Coral Calcium consisting of calcium and trace elements activates NK cells and macrophages, and plays a supportive role in inhibiting the metastasis of the cancer cells to the lungs. This inhibitory effect of Coral Calcium against the proliferation of cancer cells can be considered an effect which puts the cancer cells to sleep (does not completely kill). Accordingly, we propose to designate such effect as a “cancer-sleeping effect”.

Abstract   |   Introduction   |   Materials & Methods   |   Results & Discussion   |   References