微囊化紫草萘醌类化合物的抗肿瘤实验研究
摘要
LE体外抗肿瘤作用以MTT成色分析及形态学、DNA电泳、TUNEL和FCM分析证明。LE抑制人肝癌细胞系SMMC7721和宫颈癌细胞系Helas3细胞的增殖；这种抑制作用呈现药物剂量和作用时间的依赖性。首次报告LE对体外传代的正常细胞没有生长抑制作用，而细胞毒性药物5FU轻度抑制正常细胞：72hr培养抑制率可达30％。我们据此首次提出：LE可能是癌细胞增殖的选择性抑制剂。本论文首次证明LE对可移植性肝癌的治疗作用；也首次以试验数据显示脂质体微囊化处理的口服制剂疗效明显低于微乳液制剂。我们因而提出：在药物剂型探索中，如果用于口服给药，不宜采用脂质体剂型。本研究检测小鼠脾细胞的NK细胞毒活性、ConA诱导淋巴细胞转化以及ConA诱导的脾细胞IL-2产生三项指标，反映荷瘤和用药对小鼠免疫功能的影响。研究表明小鼠经荷瘤处理，较早时期即已出现免疫功能的抑制，表现为NK细胞杀伤活性、淋巴细胞转化率，以及IL-2产生水平均明显降低。小鼠可移植性肝癌组织切片的免疫组化染色证明：LE不仅抑制肝癌细胞的原位生长，也抑制癌细胞分泌VEGF，从而明显抑制接种局部肝癌组织内新生微血管的形成。
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 S_(180) 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.
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 S_(180) 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.
引文
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