Effects Of Monoterpenes And Mevinolin On Murine Colon Tumor Ct-26 In Vitro And Its Hepatic ''Metastases'' In Vivo

Tumors derived from the colonic epithelium exhibit cholesterol metabolism which is clearly different from that in fibroblasts, hepatocytes, adrenals, and ovaries. In hepatocytes and fibroblasts MEV inhibition of the rate limiting step in cholesterol synthesis HMG Co A reductase can be overcome by the uptake of LDL. Colon cancer cells however do not overcome MEV inhibition by LDL uptake but rather exhibit further growth suppression Mevinolin (Mevacor), a drug used to lower serum cholesterol levels has the advantage of accumulating in the liver to approximately 95% with the first pass. A small but variable percentage of non-sterol precursors may escape inhibition and be utilized for other pathways in the isoprenylation of certain proteins, among them members of the ras family. Mutated ras, an oncogene, is found in 40-50% of colon tumors and the expression of a functional gene product is dependent on isoprenylation for anchorage to the tumor cell membrane. d-Limonene, a relatively non-toxic monoterpene found in orange skin oil, selectively inhibits isoprenylation and also accumulates to some extent in the liver. It was hypothesized that the differences in mevalonate metabolism between hepatocytes and colon tumor cells could provide a chemotherapeutic advantage in which MEV and/or d-limonene could effectively inhibit cholesterol synthesis and post-translational modification of proteins with non-sterol cholesterol precursors in colon tumor derived hepatic metastases and thus inhibit their growth. Since each drug affects aspects of mevalonate synthesis at different points, the effects of the combination of their agents on inhibiting tumor metastases was investigated to ascertain if these could be additive. In tissue culture, MEV and d-limonene significantly inhibited the growth of CT-26, a murine transplantable colon tumor. Cholesterol synthesis assessed in these cells indicated that in lipid deficient media the following additions-25-hydroxycholesterol, and LDL significantly reduced cholesterol synthesis. Conversely, perillyl alcohol increased cholesterol synthesis 2.5 fold. In cells cultured in FBS based medium, which have an FBS control, MEV treatment reduced cholesterol synthesis to 65% of control. Perillyl alcohol increased synthesis 1.4 fold and when given in conjunction with MEV, it abolished the effects of this inhibitor. In isoprenylation studies of 14C-mevalonate incorporation into proteins, MEV impaired isoprenylation by restricting synthesis of mevalonate derived intermediates. Results of CT-26 treatment with perillyl alcohol are inconsistent with its putative role as a protein isoprenylation inhibitor. The combination of these agents indicates an additive action which requires additional investigation to elucidate their mechanism(s). Dietary MEV and d-limonene were evaluated alone and in combination for their chemotherapeutic potential in a hepatic "metastasis" model. Using splenic colonization in which CT-26 was implanted into the spleen and ultimately seeded the liver, each of these compounds were found to inhibit the growth of resultant tumors both alone and in combination by approximately 80% versus controls at 35 days post-implantation. Assessment of HMGCoA reductase in liver and tumor indicated that these agents were effective in reaching these target sites. The findings to date indicate that while d-limonene and MEV may differentially affect the same pathway, and their individual actions may appear antagonistic in vitro, their overall action individually or together, appears promising as a chemotherapeutic modality for the possible management of hepatic metastases from colon cancer.