| Nowadays, the incidence of lung cancer is increasing, and the trend is especially obvious in China. According to statistics, there were more than 0.65 million people died of lung cancer each year in China. At present, the clinical treatment of lung cancer is different on different types of lung cancer, SCLC is sensitive to radiotherapy and chemotherapy, and NSCLC is given priority to surgical treatment. In terms of chemotherapy, the traditional cytotoxic anti-cancer drugs could kill tumor cell, inevitably have serious side effects on kidney, liver and hematopoietic system. In order to improve the situation, this issue was based on the preliminary confirmation of Curcumol has anti-lung cancer effects, then researching the anti-cancer activity and the mechanism of curzerene, another content of zedoary turmeric. Meanwhile, synthesizing and studying antitumor activity and mechanism of ZT-21. The goal was to elucidate the resistance of Curzerene, also confirmed the anti-lung cancer effect of ZT-21 with high- efficiency. The paper was divided into two chapters: the first chapter: about the anti-lung cancer effects of Chinese herbology Curzerene, if it had positive effects, then studying its anti-lung cancer mechanisms; Second chapter: According to our previous studies of molecular imaging that arginine had targeting distribution in lung cancer, arginine was used as the targeting carrier, and coupling with the Ethyl oxalyl monochloride, then we got the Oxaloacetic acid analogues ZT-21, last evaluating its anti-lung cancer pharmacodynamics drugability index in vivo and in vitro.In Chapter I, this chapter was about Chinese medicine monomer curzerene, first, confirming its anti-tumor effect, and then studying its anti-tumor mechanisms. In order to clarify Curzerene was the anti-cancer leading compound or not. The chapter was split into six sections.In Section I, MTT assay was used to investigate the effects of Curzerene on SPC-A1 cell and MRC-5 cell. The results showed that different concentration(6.25,12.5,25,50,100 μM·L-1) of Curzerene dealing with SPC-A1 cell, the growth had been varying degrees of inhibition, its inhibition was in a dose-dependence and time-dependence pattern, using SPSS.V.11 to analyst the Curzerene inhibition rates, The IC50 of Curzerene in 24,48,72 h were 403.8 μM·L-1, 154.8 μM·L-1, 47.0 μM·L-1. But for MRC-5 cell the Curzerene had not inhibitory effect. The results suggested that Curzerene selective inhibition of lung cancer cells; it prompted its high security.In Section II, we used Hosechst 33258 cells staining method to detect the effect of Curzerene(6.25, 12.5, 25, 50, 100 μM·L-1) on apoptotic morphology of SPC-A1 cells. The results presented that, compared with the control group, the SPC-A1 cells in those groups treated with Curzerene has typical apoptosis morphological changes, also with the cells number reducing. Apoptotic cell nucleus contraction, and some of the nucleus was broken into pieces, and the color was deeper, and presented a bright blue. The results showed that Curzerene could induce apoptosis on SPC-A1 cells.In Section III, the flow cytometry apoptosis was used to study the apoptosis effect of Curzerene on SPC-A1 cells. The results showed that after 48 h treatment with Curzerene(6.25, 12.5, 25, 50, 100 μM·L-1), apoptosis rate of SPC-A1 cells increased with the concentration of the drug gradually increased, reaching 7.00%**, 8.43%**, 16.52%**, 28.21%**, 54.85%**(*p <0.05 vs. control group), respectively. Curzerene induced apoptosis of the SPC-A1 cells with a dose-dependent manner. The results were consistent with experimental results determined by MTT. It meant that large dose of Curzerene could obviously kill SPC-A1 cells; small dose of curzerene could induce SPC- A1 cells to apoptosis.In Section IV, In order to evaluate if the anti-proliferative activity of Curzerene was related to cell cycle arrest, SPC-A1 cells treated with various concentrations of Curzerene for 48 hours were analyzed using flow cytometry. The results showed that Curzerene induced cell cycle to arrest at G2/M phase in SPC-A1 cells in a dose-dependent manner, but S phase and G0/G1 phase did not present a certain dose relationship. These result suggested: Curzerene induced cell apoptosis and G2/M cell cycle arrest of SPC-A1 cells, which may be expression of its cytotoxicity.In Section V, to examine whether curzerene affect the expression of GSTA1, Real-time PCR and Western Blot were used to quantitatively analyze the GSTA1 m RNA and protein levels in each group. It was shown that both GSTA1 m RNA and protein expression levels of the cells treated with three concentrations(100, 25, 6.25 u M) of curzerene for 48 h were significantly lower than those of the control group.In Section VI, BALB/c nude mice were used as an in vivo model to assess the anti-tumor effect of curzerene. The nude mice were split into 5 groups randomly, each group had 3 males and 3 females, the 5 groups were model control, positive control, Curzerene(135 mg·kg-1), Curzerene(45 mg·kg-1), Curzerene(15 mg·kg-1). After 12 days of continuous administration, there was no death occurred to the experimental group animals. All tumor bearing mice suffered from weight gain, and the organ coefficients of liver in curzerene(45 mg·kg-1) and curzerene(15 mg·kg-1) the organ coefficients of spleen in positive control were affected, but not significant. The inhibition rates were calculated by the tumor weight, it was found that the tumors in model group grew faster than that in the treatment groups of curzerene or β-elemene. The inhibition rates for tumor weight in high, medium or low dose group of curzerene were 66.03%, 28.34% and 3.49%, respectively. While the inhibition rate of β-elemene was 49.57%. The results strongly suggested that curzerene could inhibited the tumor growth of SPC-A1 cells-bearing mice, the inhibition had dose-dependent.In Chapter II, this chapter studied the synthesis and stability of ZT-21, and then evaluated its medicinal properties. This chapter was split into four sections.In Section I, the section was about the synthesis of ZT-21. Coupling the arginine with Ethyl oxalyl monochloride by the liquid phase synthesis, and then using HPLC, MS, 1H-NMR, 13C-NMR detection means to characterized the structure of ZT-21, the results showed that the compounds obtained m/z 246, consistent with the the expected molecular weight of ZT-21. By using the method of extraction of ZT-21 purification, we got a purity of 96.56% and fluffy white solid arginine grass amide compounds ZT-21.In Section II, the section was about the initial stability of ZT-21. In this section the mainly influence factor test, accelerated test and long-term test of ZT-21 were studied. Factors affecting test: ZT-21 was placed under the high temperature(60 ℃), the high humidity(90%±5% RH, 25℃), the highlight(4500lx±500lx, 25℃) for 10 days. Respectively, on day 0, 5 and 10, the properties and content had been determined. The testing results showed that the ZT-21 under the condition of high humidity absorption of moisture condensation occurs, but stable content determination results showed that content was stable, and in the high temperature test and highlight test the moisture phenomenon is not obvious, and the content determination results showed that content was stable. Accelerated test: ZT-21 accelerating(40℃±2℃, 75%±5%RH) placed under the condition of 6 months, during the trial period, respectively 0momth, 1 month, 2 months, 3 months, 6 months at the end of each sampling time, according to the measuring method for determination of the content. The testing results showed, the ZT-21 was placed under accelerating test the content was stable after 6 months. Long-term experiment: ZT-21 was placed under room temperature(25℃±2℃, 60%±10% RH) for 12 months Respectively, on month 0, 1, 2, 3, 6 and 12, the properties and content had been determined. The testing results showed that the ZT-21 was placed under room temperature for 12 months the content was stable.In Section III, the in vivo and in vitro pharmacodynamics studies of ZT-21. MTT was used to research the effects of ZT-21 on MRC-5 cells and SPC-A1 cells in vitro. The results showed that various concentrations of ZT-21(6.25, 12.5, 25, 50,100 μM·L-1) dealing with SPC-A1, MRC-5 cells, cells growth was not obvious inhibition. In vivo, using the transplantation tumor nude mice as the model and the tumor inhibition rate as the index, using the tumor weight to calculate the tumor inhibition rate, given drug for 14 days. Experimental results showed, the ZT-21 high dose(oral), ZT-21 high dose(injection), ZT-21 middle dose(oral), ZT-21 middle dose(injection) groups of inhibitory rate were 60.38%**, 52.85%**, 39.30%*, 29.22%*, respectively. The tumor inhibition rate of cisplatin group was 66.95% **, suggesting that ZT-21 high dose group(oral) had the same anti tumor effect with cisplatin. Dosing mice weight results revealed that compared with model control group, starting from day 9, cisplatin group animals showed serious weight loss, it prompted the serious side effects of cisplatin, while 3,3,3-trifluoropropanoate group and ZT-21 groups had no markedly difference when compared with control group. That prompted ZT-21 had good security.In Section IV, the anti-tumor mechanism study of ZT-21. We used Real-time PCR and Western blot to detect the LAT1 m RNA expression and protein expression of the ZT-21 treatment nude mice tumor tissues. Experimental results presented that, when compared with control group, in ZT-21 different dosage groups’ nude mice tumor tissues LAT1 expression level were certainly difference. After comparing with the pharmacodynamics results, the tumor tissues LAT1 expression was relative to the tumor inhibition rate, the higher the dosage, the higher LAT1 expression quantity, the higher the tumor inhibition rate.To sum up, Curzerene showed anti-proliferative and apoptosis-inductive activities in SPC-A1 cells in a dose-dependent and time-dependent manner, but for the MRC-5 cells, Curzerene did not have induced apoptosis effect, prompting its selective inhibition of lung cancer cells. Curzerene could block the SPC-A1 mainly in G2/M phase, inhibited cell mitosis, and down-regulated the expression of GSTA1, then inhibited cell proliferation. Curzerene had a certain inhibition on nude mice transplantation tumor, the inhibitory rate was closely relative to the drug concentration. ZT-21 had certain anti-cancer effect, its toxicity was much lower than the positive drug cisplatin, and the anti-cancer mechanism might be related to the up regulation of LAT1. This study about the anti-cancer effect and its mechanism of Curzerene and ZT-21 could provide certain reference basis for developing clear targets and mechanisms, high efficiency, safety anti-cancer drugs. |
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