Platelet aggregation induced by adenosine diphosphate released from cloned murine fibrosarcoma cells is positively correlated with the experimental metastatic potential of the cells.

We established five clones (ML-01, ML-02, MH-01, MH-02, MH-03) from murine 3-methylcholanthrene-induced fibrosarcoma A (Meth A), and investigated their experimental metastatic potentials in relation to their platelet-aggregating activities. A clone with a high metastatic potential (MH-02) showed a characteristic biphasic pattern of platelet aggregation, of which the first peak was not present in the aggregation patterns of the clone with low metastatic potential (ML-01). The first peak was eliminated by treatment of the cells with apyrase, indicating that adenosine diphosphate (ADP) was the causative substance of this particular peak. The metastatic potential of clones correlated well with the ADP concentration of the culture media. These results suggest that the increased ADP production and consequential enhancement of platelet-aggregating activity are closely related to the increment of pulmonary metastatic potential of MH-02 clone.

Effects of intra-arterial chemotherapy with a new lipophilic anticancer agent, estradiol-chlorambucil (KM2210), dissolved in lipiodol on experimental liver tumor in rats.

Anticancer effects and biodistribution of a new lipophilic anticancer agent, estradiol-chlorambucil (KM2210), dissolved in lipiodol (LPD) were investigated as an intra-arterial chemotherapy (IAC) on Walker 256 carcinosarcoma grown in the liver of 136 Wistar rats. All rats treated with KM2210 (10 mg)-LPD survived for 90 days after administration, whereas none of the rats with LPD alone were alive for more than 19 days. Histological examination revealed that there was no viable tumor cell in the encapsulated necrotic tumor at 21 days after administration. There was no significant liver dysfunction or leukopenia due to KM2210. The biodistribution study using [14C, 3H]KM2210-LPD solution showed that KM2210 accumulated selectively in tumor and that the tumor-to-normal-liver and tumor-to-blood ratios were 10 and 1,000, respectively, at 21 days after administration. These results suggest that KM2210 has potential clinical application in the treatment of human liver cancer.

Effects of intraarterial chemotherapy using the new lipophilic anticancer agent 'KM2210' (estradiol-chlorambucil) dissolved in lipiodol on Walker 256 carcinosarcoma in Wistar rats--a preliminary report.

The effect of a new oil-soluble anticancer agent 'KM2210' (estradiol-chlorambucil) dissolved in lipiodol (LPD) was investigated as intraarterial chemotherapy, using 40 Wistar rats bearing Walker 256 carcinosarcoma in the extremities. KM2210-LPD significantly inhibited tumor growth and prolonged the survival time, as compared to LPD alone or saline alone, p less than 0.01 and p less than 0.01, respectively. Pathological study revealed that KM2210-LPD made the tumor necrotic. It was revealed that KM2210-LPD injected into the femoral artery was retained in the hind limb tumor. These findings suggest that KM2210 may possibly become a new anticancer agent with potential clinical use in LPD chemoembolization.

[Studies on platelet aggregation induced by human cultured carcinoma cell lines].

Attempts were made to clarify the mechanism of platelet aggregation and to characterize the platelet aggregating material employing established human cancer cell lines. Eleven out of the nineteen human cancer cell lines investigated showed platelet aggregating activity. The existence of divalent cation was required for the platelet aggregation induced by HMV-1 tumor cells. The platelet aggregations induced by tumor cells (HMV-1, PC-10, 3LL) were not suppressed by specific thrombin inhibitor (MD-805). The platelet aggregating activities of tumor cells (HMV-1, M 7609) were diminished by treatment with trypsin but not with collagenase or neuraminidase. Aggregating activity was preserved with a preparation of membrane from these tumor cells, although it was abolished by heating(100 degrees C 15 min) or sonication. By SDS PAGE (autoradiography), membrane proteins with MW of 20,000 daltons which specifically bound to platelets were commonly found in cells with platelet aggregating activity (HMV-1, M 7609), but were absent in platelet non-aggregating cells (HGC-25). It is therefore concluded that platelet aggregation induced by human tumor cells does not require the coexistence of thrombin, but is evoked by direct interaction of platelets with aggregating proteins (MW 20,000 daltons) on the cell membrane.

The 1 alpha-hydroxylation of 24-hydroxyvitamin D3 by chick kidney homogenates.

Tritium-labeled 24(R)-hydroxyvitamin D3 and 24(S)-hydroxyvitamin D3 were chemically synthesized and the 1 alpha-hydroxylation of these compounds by chick kidney homogenates was studied. A marked stereospecific preference with regard to the orientation of the hydroxyl functionality on carbon-24 was noted: while the 24(R)-epimer could be 1 alpha-hydroxylated in readily detectable amounts, the 24(S)-epimer was not hydroxylated. Thus, 1.2 micrograms of 1 alpha,24(R)-dihydroxyvitamin D3 was isolated and its structure confirmed by mass spectrometry. The relative rate of 1 alpha-hydroxylation of 125 nM substrate tritiated 24(R)-hydroxyvitamin D3 and 25-hydroxyvitamin D3 (the presumed natural substrate for the renal 1 alpha-hydroxylase) was 1:6.7.

Studies on the mechanism of action of 1 alpha,24-dihydroxyvitamin D3. III. The specific binding of 1 alpha,24-dihydroxyvitamin D3 to the receptor of chick parathyroid gland.

Three protein fractions of the cytosol of the chick parathyroid glands, which had the sedimentation constants of 2.5 S, 3.7 S and 5.5 S, were found to bind with 1 alpha,25-dihydroxyvitamin D3. Among these proteins, the 3.7 S protein was assumed to be the specific receptor protein. The 3.7 S receptor protein was also capable of binding to 1 alpha,24-dihydroxyvitamin D3 but not 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor protein was estimated to be 1.2 times greater than that of 1 alpha,25-dihydroxyvitamin D3, while 1 alpha,25-dihydroxyvitamin D3 bound to the receptor protein about 10 times stronger than 1 alpha,24(S)-dihydroxyvitamin D3. The dissociation constant for the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 2.7 x 10(-11) M, the dissociation constants were calculated to be 2.2 x 10(-11) M and 2.6 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3.

Studies on the mechanism of action of 1 alpha, 24-dihydroxyvitamin D3. II. Specific binding of alpha, 24-dihydroxyvitamin D3 to chick intestinal receptor.

The binding of vitamin D3 analogues to the chick intestinal cytosol receptor was studied. In intestinal cytosol fraction, receptor proteins having the sedimentation constant of 2.5 S and 3.7 S to which 1 alpha,25-dihydroxyvitamin D3 binds were present, and the latter was specific for the compound. The binding of 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3 to the receptor was also observed, while very weak binding was seen in the case of 24(R)25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor was 1.3 times as high as that of 1 alpha,25-dihydroxyvitamin D3, whereas those of 1 alpha,24(S)-dihydroxyvitamin D3, 1 alpha-hydroxyvitamin D3 and 25-hydroxyvitamin D3 were 10, 304 and 652 times lower than 1 alpha,25-dihydroxyvitamin D3, respectively. The dissociation constant of the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 3.0 x 10(-11) M, and the dissociation constants were calculated to be 2.4 x 10(-11) M and 2.7 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3, respectively.

Studies on vitamin D metabolism: catabolism of 1 alpha, 25-dihydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 to a metabolite inactive on bone mineral mobilization.

A heretofore unknown metabolite of vitamin D3 was isolated from the 1 alpha, 24, 25-trihydroxyvitamin D3 fraction of lipid extracts obtained from plasma of rats which were given intravenous or oral doses of 100 pmol/100 g of either 1 alpha-hydroxyvitamin D3 or 1 alpha, 23-dihydroxyvitamin D3. Doses of 25-250 pmoles of the new metabolite when given to a vitamin D deficient rat were completely inactive in terms of stimulating the classic vitamin D response of bone calcium mobilization. The nature of the metabolism of 1 alpha-hydroxyvitamin D3 or 1 alpha, 25-dihydroxyvitamin D3 to the metabolite is not clear at the present time, but it is probable that neither of these steroids undergo side-chain cleavage to yield the new metabolite.