The Experimental Study On Anti-carcinoma Effects Of Shikonin Derivatives

Naphthoquinones are the major pharmaco-active components of a Chinese medical herb, Zicao. their derivatives, designated as LE, are isolated from the plants and made water-soluble, whereby to analyze their anti-carcinoma pharmacokinetics both in vitro and in mouse models.MTT assay was conducted to examine the anti-carcinoma actions of LE, with 5FU as the positive control and with HEK293 and NIH3T3 as the normal cell control. Both the drugs were exhibited to suppress growth of human hepatoma cell line SMMC7721 and cervical cancer cell Helas3 in vitro. Some differences, such as the sensitivities of tumor cells and the action intensity, were observed between LE and 5FU, indicating different mechanisms for the pharmaco-activities of the 2 kinds of drugs.Distinguished from that of 5FU that inhibited in vitro growth of the normal cell lines in 72hr co-culture with up to 30%, LE did not show any influences on the propagation of either HEK293 or NIH3T3. On the basis of searching in Pubmed till 2007 and Wanfang database till 2006, LE was firstly reported as an agent that did not interfere growth of normal cells.The mechanisms responsible for cancerous inhibition of LE were investigated with morphology, genomic DNA electrophoresis in agarose gel, and with TUNEL analysis. With the maximal dosage of 10μg/ml in our experiments, HEK293 cells were in their normal situation in presence of LE, observed under light microscopy and electronic microscopy. There was no degradation of genomic DNA of the cells in presence of LE, even during the incubation of up to 72hrs. On the other hand, SMMC7721 was shown in a typical process of apoptosis during 72hr co-culture with LE: the chromatin condensation at 24hrs; the so-called wheel-like appearance of nucleus at 48hrs, resulted from attachment of condensed chromatin beneath the inner membrane of the nucleus; and formation of apoptotic body under electronic microscopy. It was observed that not only apoptosis, but also necrosis of the tumor cells occurred in 72hr culture in presence of either LE or 5FU. It is therefore concluded that it may be the normal process of cells undergoing apoptosis so that it is not surprising to find the necotic profile of the cells in agarose eletrophoresis.We found that handicap of DNA synthesis might be the step prior to apoptosis of the tumor cells when cultured with LE, which was indicated by the FCM examination for the 16hr culture in presence of the drug. As the result, it is supposed that it is the inhibition of DNA synthesis by LE lead to the cells going to apoptosis and then to necrosis.The drug was given by gavage to the mice inoculated with either Hep-A-22 or Siso sarcoma cells, at dosage of 2.5-10mg/kg daily for 10 days. Survival times of the tumor bearing mice and the tumor sizes were scored. The organ indexes of both thymus and spleens were detected and the organ underwent pathological analyses. Immune functions including NK cytotoxicity, ConA-induced lymphocyte transformation as well as the IL-2 production were analyzed. The survivals of tumor bearing mice in all drug-treated groups (T) were much more prolonged than that of the control group (C) with T/C ratio beyond 140% (143-157%). The in situ propagation of dorsal tumor was obviously inhibited by the drugs under the given doses (P<0.001), which was indicated by tumor mass measured at the 14^th day after inoculation with tumor cells. The suppressed VEGF expression and its consequence of diminished MVD were probably one of the reasons for delayed proliferation of the inoculated tumor cells. At the same time, the tumor-bearing mice in control group without drug treatment became in the condition of dyscrasia and application of 5FU resulted even in the worse somatoplasm.It may be the first report that involved with the morphological examinations of the tumor-bearing mice. Thymus and spleen atrophy of the tumor-bearing mice were exhibited in the H-E staining slices. Thymus atrophy was presented in the thymic cortex, identified by disappearance of the cortex. It was astonished that the pathological changes in the spleens of the tumor-bearing mice were mainly discovered in the secondary nodules. Besides those mentioned above, the immune functions examined were significantly decreased in the mice inoculated with either Hep-A-22 or S₁₈₀ carcinomas.Noticibly, the regimen of LE ameliorated the systemic conditions of the tumor-bearing mice. Both the organ indexes and the morphology of thymus and spleen were amended in tumor-bearing mice receiving LE treatment. And all the criteria for immunobiology were found improved in the mice. The immunological parameters of the mice were even found to be upregulated due to application of LE.Conclusion: LE may be a promising therapeutic agent in regimens for cancerous diseases.