The Anticancer Effects And Related Mechanism Of 3-Bromopyruvate

Tumor cell's energy is mainly from glucose oxidizing and there is a great difference between the tumor cell and normal cell in glucose metabolism .The main character of tumor cell is that glycolysis increases dramatically, and the more increase obviously, the more malignant it is. 3-bromopyruvate (3-BrPA) is a representative of glycolysis enzyme inhibitor whose target is glycolysis rate-limiting enzyme which is progressing rapidly on anticancer researches, especially on inhibiting hepatoma. As an anticancer agent,the research on 3-BrPA is just on primary level and the range of its antitumor research is limited. Its anticancer effects and its adverse effects have been observed by inhibiting proliferation of hepatoma cell, intervenient administration on liver cancer animal model and injecting directly on cancer-bearing animals. All of these researches about its antitumor mechanism are mainly on energy metabolism of glycolysis. How 3-BrPA acts on other tumors, whether it exists other antitumor mechanisms and what adverse effects may appear after multiple administrating are rarely reported abroad, and its antitumor effects in vivo and in vitro have never been reported internally.In our study, the anticancer effects of 3-BrPA in vitro have been examined by modern cytobiological, molecular biological, pathobiological and toxicological analyses based on the achieved researches. Moreover, to find its further anticancer mechanism, the inhibitory effects on the cell ultramicrostructure,cell cycle and gene expression of MCF-7 and SWⅢ-C have also been investigated. To observe adverse effects and evaluate its safety 3-BrPA are administrated by different ways. Our studies may offer theory and experiment evidences for researching and exploring of 3-BrPA which is promising to be a new anticancer agent.First 1 The anticancer effects of 3-BrPAObjective: Screening and Evaluating of anticancer spectra of 3- Bromopyruvate in vitro.Methods: To detect nine kinds of human tumor cell in vitro by MTT methods.Results: It shows that 3-BrPA has inhibitory effects on nine kinds of human tumor cell in vitro by MTT methods. Its IC50 is from 6.44μg/ml to 20.78μg/ml. The IC50 of 3-BrPA on SWⅢ-C,MCF-7 and U125 cells are all less than 10μg/ml.Conclusions: It is demonstrated that 3-BrPA has wide spectra of anticancer in vitro. Part 2 The anticancer mechanism of 3-BrPAObjective: To investigate the effects of 3-BrPA on cell proliferation, cellular morphology, the ultramicrostructure, cell cycle distribution, apoptosis rate, and bcl-2,c-myc and mtp53 protein expression of SWIII-C and MCF-7 cells.Methods: The effects of 3-BrPA are examined by electron microscope methods and MTT.Results: (1)Effects on cell proliferation of MCF-7 and SWⅢ-C. The results show that 3-BrPA inhibiting cell growth on MCF-7 and SWⅢ-C which shows time dependence and dose dependence under certain dose range. There are conspicuous inhibiting effects on MCF-7 and SWⅢ-C when the concentration of 3-BrPA is between 25μg/ml and 100μg/ml.The most obvious inhibiting effect is that when it is at 25μg/ml, it will decrease as the concentration increasing.(2)Effects on cellular morphology and ultramicrostructure of SWIII-C and MCF-7. Under conventional culture, 3-BrPA (25μg/ml) is added respectively, and then cells are collected, fixed, embedded, made slides, stained and observed by transmission electron microscope. The morphological characteristics of normal cell under electron microscope are that the morphous and structures are regular with regard to its uniformly distributed chromoplasm and integrated cellular membrane. After treated with 3-BrPA 48h, apoptosis cells present karyopycnosis phenomenon, which chromatin congregates beside Cary theca like crescent with different shape and size , cytoplasm brimming particles and vacuoles as well as limpid boundary and integrated cellular membrane.(3)The impact on cell cycle distribution and apoptosis rate of SWIII-C and MCF-7. Under conventional culture, treated with 3-BrPA for 48h respectively, collecting cells and staining by PI, analyzing cell cycle phase distribution and apoptosis rate by flow cytometry. The results demonstrates that except low concentration of 3-BrPA apoptosis have been induced by 3-BrPA between 25μg/ml and 100μg/ml and the most prominent effect is at 25μg/ml. Analyzed cell cycle shows that compared with control group, accompanied by the proportion of G2/M increasing the proportion of S and G0/G1 in SWIII-C decrease as the concentration of 3-BrPA elevating. After treated with 3-BrPA, cell cycle of MCF-7 displays that as the percentage of G0/G1 decreases, both the percentage of S and G2/M increased, we can conclude that 3-BrPA arrests tumor cell proliferation on M and G1 to induce apoptosis.(4)The impact on bcl-2,c-myc and mtp53 gene protein expression of SWIII-C and MCF-7.Under conventional culture, disposed t with 25μg/ml of 3-BrPA for 96h, then collecting, filming, SP staining, microscope examining and analyzing data. The result of immunohistochemistry is that there are inconsistently buffy substance like particles in cytoplasm and nuclear. The expressions of Bcl-2,c-myc and mtp53 proteins were lowered by 3-BrPA. It is explained that 3-BrPA has down regulated bcl-2,c-myc and mtp53 protein expression in tumor cells.Conclusions: (1)3-BrPA can obviously inhibit the multiplication of SWIII-C and MCF-7 cells. The strongest inhibition is that of the concentration 25μg/ml(.2)3-BrPA mainly arrests G2/M and G1 to induce apoptosis which is closely relative to gene protein expression of bcl-2,c-myc and mtp53. Part 3 Safety evaluation of 3-BrPAObjective: To evaluate the safety of 3-BrPA. Methods: The safety of 3-BrPA was examined by pathobiological and toxicological analyses.Results: (1)The influence on experimental animals. Experiment animals'toleration to 3-BrPA is fine and the damage of local injection is trivial.(2)The influence on hematological system and serum parameters. Rats are injected 3-BrPA (0.55mg/kg) via tail vein once a day, after a period of 7 days, blood samples are collected and serum parameters are detected. The control rats serve as saline estimated bad general condition and hypocytosis accompanied by the content of ALT,Urea,Glob,γ-GT increasing and Glu decreasing.(3)The influence on histomorphology of rabbits. Rabbits are intravenously injected 3-BrPA by different administration way and dosage. The first mode is that single intravenous injection of 3-BrPA(1.1mg/kg), 7 days after collected samples. The second mode is that intravenous injection is done once a week (0.55mg/kg), 4 weeks after collected samples. Positive control rabbits are treated with Cisplatin by same method at two dosages (4mg/kg; 2mg/kg) and normal control rabbits are administrated by physiological saline. We discover that the group treated by the first mode is in well condition. Two rabbits treated with Cisplatin die and the weight of other rabbits decreases. Except that one rabbit treated by the second mode die at third week, others are all well. Histomorphological analysis of rabbit treated with 3-BrPA presents that except liver and kidney have some changes, other organs like thoracic gland, spleen, heart, pancreatic gland, adrenal gland, stomach, dodecadactylon, hy stera and didmus have no morphological change. Compared with the positive control group treated with Cisplatin (4mg/kg), in the group treated with 3-BrPA by the first mode, the probability of hyperemia of glomerular capillary decreases from75% to 66.7% and the probability of hydropsia of hepatic cells cytoplasm decreases from 50% to 33.3%. Among the group treated with 3-BrPA by the second mode, the probability of hyperemia of glomerular capillary is 60% and the probability of hydropsia of hepatic cells cytoplasm is 100%. Comparatively, among the group treated with Cisplatin (2mg/kg), the probability of hyperemia of glomerular capillary is 83% and the probability of hydropsia of hepatic cells cytoplasm is 50% and one of them characterizes as chronic hepatic inflammation. Compared with the normal control group and 3-BrPA treated group, 50 % of Cisplatin group presents fewer layers and less density of spermatogenesis cells in testicle.(4) The influence on haematogenesis of rabbits. According to bone marrow examination, the group with single injection of 3-BrPA at high dosage shows that 33.3% is normal, 50% indicating slight bone marrow depression with actively accreted nucleated cells and senile remarkably cells, 16.7% appearing bad bone marrow depression with the decreased nucleated cell and hematopoietic cell, as well as the increased fibrocyte. Accordingly, in Cisplatin group, 75% is normal, 25% indicates bone marrow depression with the decreased nucleated cells and hematopoietic cells as well as the increased fibrocyte. The group treated with small dosage of 3-BrPA once a week and lasting 4 weeks, the bone marrow expression is that 60% characterizes as actively accreting nucleated cells , widely appearing hematopoietic cells, obviously fat vacuoles and singular megacaryocytes; 40% is badly depressed bone marrow expression. In Cisplatin group, 83% is badly depressed bone marrow and 17% characterizes as actively accreting nucleated cells and increased hematopoietic cells and singular megacaryocytes. (5) The influence on hematological system and hepatic and renal function of rabbits. There is insignificant influence on the amount of RBC, WBC and Hb by two different administration modes except that the quantity of PLT has been obviously decreased, especially when large dosage administrates at one time. As 3-BrPA accumulates, rabbits'liver and kidney are damaged gradually and the serum content of ALT and Urea has gradually increased.Conclusion: (1)Experiment animals'toleration to 3-BrPA is well; the damage of injection site is trivial. (2)There is difference between two administration modes on the hematological system of experimental animals. It leads blood cells seriously decreased when administrated by low dosage for one week. Both large dosage at one time and low dosage for 4 weeks induced PLT in peripheral blood apparently decrease, and the former mode is much more obvious than the latter one. (3)As 3-BrPA accumulates, the contents of ALT and Urea in blood serum increase accompanied by animals'liver and kidney damaged gradually and renal function has injured badly.(4)Although some rabbits'liver and kidney have some morphological changes, there is no abnormal morphological changes on thoracic gland, spleen, heart, heart, pancreatic gland, adrenal gland, stomach, dodecadactylon, hystera and didmus under two administration mode of 3-BrPA.(5)Under two administration modes of 3-BrPA, some rabbits'bone marrow expression show haematopoiesis inhibition, and megacaryocyte is inhibited badly.Conclusion:1. 3-BrPA has wide spectra of anticancer effects in vitro and its effect is very significant.2. 3-BrPA can obviously inhibit the multiplication of SWIII-C and MCF-7 cells. The strongest inhibition is that of the concentration 25μg/ml. 3-BrPA mainly arrests M and G1 to induce apoptosis which is closely relative to gene protein expression of bcl-2,c-myc and mtp53.3. The experiment of the chronic toxicity of 3-BrPA in rats and rabbits indicate that 3-BrPA is relatively safe.