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Experimental Study of the Antiproliferative Activity of Melatonin on HeLa Cells and Its Related Effect

Author: ChenShaoYa
Tutor: ChenChongHong
School: Fujian Medical
Course: Internal Medicine
Keywords: Melatonin(MT) HeLa cells Antiproliferative activities NFκB IκB-α p-IκB-α IκKα/β Cell division cycle Phase S T-AOC
CLC: R737.33
Type: PhD thesis
Year: 2007
Downloads: 156
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Abstract


Melatonin(MT) is the indole hormone widely existing in organism. It possesses some important physiological and pharmacologic properties, such as enhancing body immune function, elevating the antioxidative activities and antitumor activities. Furthermore, melatonin has very low toxicity. Therefore, the study on function of melatonin has been one of the most interesting research fields.It is one of the focuses that MT can be a chemopreventive drug and antineoplastic activities and its prospect of application. The death rate of metaphase or advanced cervical carcinoma has kept on high level. After surgical operation, the patients suffered from metaphase or advanced cervical carcinoma are necessary for the chemotherapy. Studies showed that the plasma MT concentration in patients with cervical cancer was lower than that of normals. Therefore, it is logical to assume that MT may be help to cervical carcinoma patients.In the present study, the antineoplastic effects of melatonin determined by cultured cell lines and animal transplanting tumor models in order to make clear further the antineoplastic effects of melatonin. The expression of NF-κB and its regulatory proteins by MT on HeLa cells were analysied primarily by Western blotting analysis. Then, the study was designed to evaluate the influence of melatonin on the cell division cycle of HeLa cells, to explore morphological alterations of HeLa cells by fluorescene staining and transmission electron micrograph. In addition, the T-AOC and the level of T-SOD and GSH on HeLa cells by MT would be measured. The paper would detect the MT membrane receptors MT1 and MT2 on HeLa cells by means of Western blotting analysis and immunohistochemical staining.These study contributed to a better understanding of the antineoplastic activities and its related effects which were undertaken to supply the experimental foundations for the potential clinical application of melatonin and its homologens as a kinds of antineoplastic drugs or adjuvants in treatment of neoplasms.1 Antineoplastic activities of MTThe cell growth curve of HL60, B16, HeLa and MCF-7 were determined by MTT assay. With the elevation of dose and accession of time, the antiproliferative activities induced by MT increased. All of the cell growth curve were significantly lower. The IC50 on HeLa cells which was the smallest one of the four cell lines by MT for 72hr was 61.71μg / mL. The above results have been confirmed further that MT had an antiproliferative activities on cultured tumor cell lines in vitro.It was shown that MT inhibited the murine U14 carcinoma in a dose-dependent manner with the maximum tumor growth inhibitory rate of 30.9% at a dose of MT 80mg / kg. It was also shown that the means of body weight either MT group or MT in combined with CTX or 5-FU were gain more than that of both control and CTX group. It confirmed that MT had no obvious toxicity. The results observed that both of MT in combined with CTX (q=1.072)and with 5-FU(q=1.093) were showed synergistic. These results suggested that melatonin possesses an antineoplastic activities in vivo.2 The influences of MT on the levels of NFκB and its related proteins IκB-α, p-IκB-αand IκKα/βon HeLa cellsIt was shown that MT could down-regulate NFκB protein level in a time- dependent and dose-dependent manner after treated with melatonin 0.2μg /μL for 1d, 2d and 3d and with melatonin 0.008μg /μL, 0.04μg /μL and 0.2μg /μL for 3 days by Western blotting analysis. It was observed that MT could down-regulate IκKα/βand p-IκB-αand up-regulate IκB-αafter treated with melatonin 0.2μg /μL for 1d, 2d and 3d in a time-dependent manner from the second day. The results revealed that MT could down-regulate IκKα/βand p-IκB-αand up-regulate IκB-αafter treated with melatonin 0.008μg /μL, 0.04μg /μL and 0.2μg /μL for 3 days in a dose-dependent manner from MT 0.04μg /μL. These results suggested that MT could down-regulate NFκB protein level while exerting the antiproliferative activities. Moreover, These results also suggested that MT regulated NFκB related proteins level, including down-regulate IκKα/βand p-IκB-αand up-regulate IκB-α. These may be result in inhibiting NF-κB activity.3 Other effect of MT related to its antiproliferation activitiesIt was observed that HeLa cells stained with AO/EB were damaged in a time-dependent manner by fluorescene staining. As the time go on, more and more necrocytosis were seen and less apoptosis by MT 1μg /μL for 24h, 48h and 72h.The results showed more somatic chromosome and less hetero-chromosome of the control group. It was observed that much more necrocytosis and less apoptosis presented on HeLa cells exposed to MT 0.2μg /μL for 72h by transmission electron micrograph.The results also showed that HeLa cells in phase S was reduced in a dose-dependent manner while exposed to melatonin at 0.008μg /μL、0.04μg /μL and 0.2μg /μL for 72h by flow cytometric analysis.It was shown that MT enhanced T-AOC in a time-dependent and dose-dependent manner on HeLa cells after exposed to melatonin at 0.008μg /μL , 0.04μg /μL, 0.2μg /μL and 1μg /μL for 1d, 2d and 3d by uv-vis spectrophotometer.MT elevated the level of T-SOD(dose and group as the same as above mentioned) and GSH(dose and group similar to above mentioned, not including the very high group MT) on HeLa cells. The results showed that MT1 and MT2 MR were not be detected on HeLa cells by Western blotting analysis and immunohistochemical staining. Whether the MT nucleus receptors ROR/RZR existed on HeLa cells and participated the antiproliferation activities of MT were still unknown and need to be further confirmed.These results above mentioned revealed that MT inhibited HeLa cells with necrosis and less apoptosis, along with reducing the cell numbers of phase S in the cell division cycle, enhancing the T-AOC and increasing the level of T-SOD and GSH. These rusults determined that MT1 and MT2 MR were not be detected on HeLa cells and MT nucleus receptors ROR/RZR existed or not were still unknown and need to be analysed.Summary1. It have been confirmed further that MT 0.008μg /μL-1μg /μL had an antiproliferative activities on HeLa cells in vitro. The IC50 of MT on HeLa cells was 0.617μg /μL.2. MT 80mg / kg possesses an antineoplastic activities on murine transplant carcinoma U14 and had no obvious toxicity in vivo. The maximum inhibitory rate on U14 was 30.9 %. Both MT in combined with CTX (q=1.072)and with 5-FU(q=1.093) were showed synergistic.3.MT could down-regulate NFκB protein level while exerting the antiproliferative activities, which may be one of mechanisms of the antiproliferative activity of it.4. MT regulated NFκB related proteins level, including down-regulate IκKα/βand p-IκB-αand up-regulate IκB-α. These may be result in inhibiting NF-κB activity, which may be contribute to the antiproliferative activity of it.5.MT 0.008μg /μL - 1μg /μL enhanced T-AOC in a time-dependent and dose-dependent manner on HeLa cells. MT elevated the level of T-SOD and GSH, which may be related to the antiproliferative activity of it. 6. HeLa cells in phase S was reduced by MT 0.008μg /μL - 0.2μg /μL for 72 hr, which may be also related to the antiproliferative activity of it.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Uterine tumors
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