In 1930, Ishihara extracted an anticancer substance from the placenta and fetal umbilical cord. In 1934, Murphy and Strum reported a definite inhibitory action of extracts of placenta on the growth of transplantable cancers of mice and observed that extract of desiccated homologous embryo skin and placenta markedly decreased the rate of postoperative local recurrence after surgical removal of spontaneous cancer of mice. In 1958, Chin and Jhun demonstrated the growth inhibitory effect of emulsion or aqueous extract of placenta on mouse ascites cancer. On the other hand, growth inhibiting substance were obtained from various tissues or tumors by numerous investigators (Marshak and Walker, Stich and Florian, Herbut, Doljanski, and Nutini.)
Chin, B.H. and Kim, C.S. extracted tissue factors especially from the liver of white rat and demonstrated suppressive or even regressive effect on the growth of Ehrlich ascites cancer and N-F sarcoma.
This work concerns with attempts to investigate these aspects further and to study the presence of growth inhibitory substances in human planenta by the methods essentially similar to those used by Chin, B. H. and Kim, C.S.
Materials and Methods
Ehrlich ascites cancer and N-F sarcoma(Nakahara-Fukuoka) were used. Ehrlich´ ascites cancer cells were aspirated and diluted with saline so that each 0.1 ml contains approximately one million cancer cells. One tenth ml of the preparation was inoculated subcutaneously.
Tissue factors were prepared from human placenta (3-9 months) -in the following manner. The placentas were minced into pieces with scissors and four volumes of distilled water per volume of minced tissue were added. The mixture was then emulsified in a Waring blender. The emulsion was extracted for several hours at room temperature, and then filtered through three ;layers of fine mesh gauze.
The resulting filtrate was sedimented in Model PRI International Centrifuge at 15>000/r.p.m. For 40 minutes. The supernatant was then removed and 1-2 volumes of _95 percent ethyl alcohol added to this supernatant and the mixture was kept in refrigerator for 24 to 48 hours.
The resulting precipitate resuspended in a small amount of distilled water was subjected to paper electrophoresis in sod. barbital and acid barbital buffer system at pH 8.6. Slow moving fractions cut on 3 cm from the spotting, point were elluted with distilled water.
The eliuent was subjected to centrifugation and the sediment was dried in a vacuum desicator.
To, this an appropriate amount of distilled water was added to make a 10 per cents suspension. Then the suspension was treated with trypsin in 37° C incubator at pH 8 for 24 hours. The solution was transferred into a cellophane tube and dialyzed against distilled water in refrigerator for 72 hours.
In some cases, a magnetic stirring machine was used for dialyzation. The substances in the cellophane tube was removed and precipitated by adding the same volume of acetone. The sediments were dried and the resulting powder was ready for injection as, 1 or 2 per cent solution in distilled water. The injection started on the day of tumor transplantation in mice. The daily doses per animal consisted of 0.1 and 0.2 ml each of 1 per cent solution and 0.2, 0.3 and 0.4 ml each of 2 per cent solution.
The size of the tumor was directly measured from the skin with calipers. One half of the values of the largest length and largest width as measured on the line vertical to the former was considered as the- index of the size of tumor. On the 18th day of injection, animals were sacrificed and tumors removed and weighed.
Results
As shown in Table 3, 4, 5, and 6, in the animals injected with 0.2 and 0.4 ml each of 1 per cent solution and 0.2 nil of 2 per cent solution, the growths of ascited cancer(solid form) and N-F sarcoma were inhibited, the degree of inhibition being apparently more marked in ascites cancer than in N-F sarcoma.
In animals injected with 0.3 ml of 2 per cent solution, the growth was completely inhibited by the 12th day of injection, but the mortality rate increased even though the tumor growth became stationary.. The tumor removed on the 18th day showed a marked decrease in weight, particularly those of. the group injected with 0.3 ml of the 2 per cent solution.
As shown in Table 8, in animals injected with 0.4 ml of the 2 per cent solution, the inhibition of growth was marked by, the 9th. day of injection and occassional regression was observed thereafter.
The toxic effect of this factor was studied by administering the material to normal mice. As can be seen in Table 2, some animals injected with 0.3 and 0.4 ml each of 2 per cent solution died at various intervals after injection, but injection of 0.2 and 0.4 ml each of 1 per cent solution did not exert lethal effect in mice.
The death rate after the injection of tissue factor was greater in tumor-bearing animals than in normal animals. The injection of, 0.3 and 0.4 ml each of 2 per. cent solution of tissue factor resulted in some loss of body weight in normal mouse.
From the results obtained, it can be concluded that the presence of cancer growth inhibitory factor in placenta is. Apparent, The chemical nature of tissue factor and the observation that toxic effect was severer in cancer animals than in normal animals may require further investigation.
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