| ObjectiveTryptanthrin, 6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline, is a kind of indole quinazoline alkaloid. It is a yellow needle crystal and its melting point is between 267 and 268 centidegree. Tryptanthrin was isolated firstly from plants such as Strobilanthes cusia, Polygonum tinctorium Lour and Isatis tinctoria L. Literatures reported that tryptanthrin possessed anti-bacteria, anti-fungus, anti-inflammation, anti-tumor, anti-parasite activities and so on. But up to now, the researches of tryptanthrin mainly focused on the aspect of separation, extraction and synthesis because of the limited resource. The pharmacological activity studies of tryptanthrin mainly focused on anti-inflammation and little on anti-tumor. Morever, the study of tryptanthrin on hematological system tumor was only on acute promoyelocytic leukemia cells in vitro, there still has not the reports of proliferation, apoptosis and cell cycle effect of tryptanthrin on human chronic myeloid leukemia cell line-K562. In the study, tryptanthrin, synthesized by biomimetic synthesis method, was taken as study object to investigate its inhibitory activities and the action mechanism on human leukemia cell line-K562 adopting the neoplastic cells cultured in vitro and animal model bearing tumor in vivo, which provide the experimental data for the clinical application of tryptanthrin.MethodsPart One: the anti-tumor effect of tryptanthrin on eight kinds of tumor cell and the study for tryptanthrin on inhibiting proliferation, inducing apoptosis of K562 in vitro By means of inhibiting proliferation assay, the anti-tumor sensitive activities of tryptanthrin were screened primarily on eight kinds of tumor cells including A549, HepG2, SW480, Eca-109, GRC-1, LNCAP, A375, K562. The tumor cell which is the most sensitive to tryptanthrin was ascerted as target cell. The concentration and time of tryptanthrin on the tumor cell were ascerted. The tumor cell in logarithmic growth phase was added tryptanthrin with different concentration to incubate for indicated time and then some indexes were detected to observe the change of morphology by contrast phase microscope, Hoechst 33258 fluorescent staining and transmission microscopy, analysis the cell apoptosis rate with Annexin-V-FITC and PI double labelings, and the change of cell cycle with PI simple staining by flow cytometer.Part Two: the action mechanism of tryptanthrin on K562 cell in vitro The mitochondrial transmembrane potential was determined using Rhdamine 123 fluorescein stain by flow cytometer. After treated with tryptanthrin, the apoptosis relative gene expression of Bcl-2, Bax mRNA, caspase-3 and fusion gene bcr/abl mRNA in K562 cells were detected by SYBR Green Realtime PCR method. The expression of apoptosis relative protein Bcl-2, Bax, cytochrome c, caspase-3 and fusion protein bcr/abl were detected by Western blot.Part Three: the treatment effect and action mechanism of tryptanthrin on leukemia-K562 mouse Combining with the anti-tumor effect data of tryptanthrin in vitro, the anti-tumor action mechanism of tryptanthrin in vivo was assessed using tumor-bearing mice SCID mouse. The general leukemia animal model was established adopting cytoxan pretreatment and vena caudalis injection with 8×106 K562 cells per mouse. After intragastric administration with 50mg/kg and 25mg/kg tryptanthrin for 14 days, the therapeutic effects of tryptanthrin were assessed by the change of body weight of animals, peripheral blood and bone marrow smear, leukocyte differential counting, the expression of bcr/abl mRNA, the analysis of cell cycle, the expression of cell surface antigen CD13, the expression of fusion gene bcr/abl mRNA. Meanwhile, the bone marrow, heart, liver, spleen, lung and kidney were taken to pathological section with haematoxylin and easin staining.ResultsFirst, the inhibitory effect of tryptanthrin on tumor cells MTT assay results confirmed that tryptanthrin was the most sensitive on K562 cell among eight kinds of tumor cells and could inhibit significantly on proliferation of K562 cells in a concentration and time manners. The cell morphous and proliferation cycle could be changed by tryptanthrin after 48 hours. At dose of 12.5μg/ml and 25μg/ml tryptanthrin, the cell density was decreased obviously and the morphologic change of K562 cell was altered significantly by contrast phase microscope, which exhibited ruptured cellular membrane, weaken brightness and even dissolved death of cells. Transmission electron microscopic results showed that the apoptosis cytosis, karyopyknosis, chromatin margination to crescent or circularity and cellular necrosis. Staining with Hoechst 33258 for fluorescence microscopy, some of the tryptanthrin-treated cells exhibited highly condensed and fragmented nuclei morphology, which were the typical characteristics of apoptosis. In contrast, the cells in the culture without tryptanthrin and dissolvent group respectively showed normal cell nuclei morphology with diffusion and uniform fluorescent light. The flow cytometer results indicated that tryptanthrin could not only induce apoptosis, but also influence the cell cycle. The normal cells decreased obviously, the proportion of apoptotic cells and necrotic cells increased significantly after treatment for 48 hours. With the concentration raised, viable apoptotic cell decreased and the proportion of non-viable apoptotic cell and necrotic cell increased obviously. The changes of cell cycle were obvious and the proportion of S phase decreased less than that of control group. Especially at the dose of 25μg/ml, the percent of S phase decreased to 33.2% (P<0.01). The cell population of the G0/G1 phase and G2/M phase was significantly increased up to 52.6% and 14.3% (P<0.05, P<0.01).Second, the inhibitory effect mechanism of tryptanthrin on K562 cells Cells were treated with different concentrations of tryptanthrin and stained with Rhodamine 123 after 48 hours. Mitochondrial membrane potential of K562 cells decreased in a dose-dependent manner, which was consistent with the result of cell apoptosis. Western blot results indicated that the protein expression of Bcl-2 was downregulated and the expression levels of Bax were upregulated gradually after treatment with 6.25, 12.5 and 25μg/ml tryptanthrin. With the concentration raised, the expression of cytochrome c in cytoplasms and caspase-3 in cells were both increased. In addition, the expression of bcr/abl protein can be inhibited by tryptanthrin at the dose of 25μg/ml. The mRNA expression levels of Bcl-2, Bax, caspase3 and bcr/abl by SYBR Green PCR were in accordance with the protein expression levels by Western blot.Third, the establishment of leukemia SCID mouse and the evaluation of therapeutic effect of tryptanthrin on leukemia SCID mouse The leukemia SCID animal model was established successfully adopting to cytoxan pretreatment and vena caudalis injection K562 cells, which exhibited the weight loss and wasting disease. The amount of peripheral white blood cell had a marked increasing after injection three weeks. Myelocyte and metagranulocyte could be observed in peripheral blood and bone marrow smear. After treatment with 25, 50 mg/kg tryptanthrin and 180 mg/kg hydroxyurea, the general state and the body weight of mouse was improved. The amount of peripheral white blood cell decreased. The FCM results showed that the expression of CD13 of bone marrow in tryptanthrin treatment group was decreased significantly compared with leukemia SCID group (P<0.01). The proportion of S phase of bone marrow cells was decreased. The pathology results indicated that the inflammation actions in organs induced by leukemia cells were improved to some extent. The therapeutic effect of 50 mg/kg tryptanthrin was better than that of 25 mg/kg tryptanthrin.ConclusionsWith fluorescein stain, transmission microscope and flow cytometer methods, the inhibitory effect of tryptanthrin was confirmed on K562 cell in vitro and leukemia induced by K562 cells in vivo. The action mechanism of tryptanthrin might on the one hand, regulate the expressions of Bcl-2, Bax mRNA and protein, degrade the mitochondrial transmembrane potential, make the cytochrome c release to cytoplasm and activate the downstream molecular caspase 3, on the other hand, inhibit the expression of bcr/abl mRNA and protein to suppress the proliferation and induce apoptosis of K562 cells. |
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