The Role Of Pin1 In Carcinogenesis And Development Of Malignant Melanoma The Establishment Of Human Peptidyl-prolyl Cis-trans Isomerase Inhibitor Screening Method And Study Of The Mechanism Of Action The Antitumor Activity And Mechanisms Of XLN306

Malignant Melanoma (MM), a cancer of melanocytes, pigment-producing cells in the skin, is the most serious form of skin cancer and its incidence has been rising steadily throughout the past several decades. It is considered curable when detected at early stage but once entering the advanced-stage, it will disseminate widely and soon become an incurable malignancy with extremily poor prognosis. Until now, there has been no systemic and effective therapy that has clear effect on overall survival of patients with MM. Therefore, a penetrating understanding of the pathogenesis of MM and search of potent molecular targets based on the etiology of MM may lead to effective strategies in the treatment of this cancer.The conformational regulation of phosphorylated Ser/Thr-Pro motifs in a certain subset of proteins is a novel signaling mechanism after phosphorylation pivotal in regulation of many cellular functions, especially proliferation and transformation. The key enzyme—peptidyl-prolyl cis/trans isomerases Pinl can specifically recognize and isomerize the phosphotylated Ser/Thr-Pro bands in certain proteins, thereby changing their conformation and modulating their functions. Recent work indicated that Pinl was overexpressed in many human cancers and could promote many oncogenesis signaling as an "oncogenic catalyst"Bao et al. showed that Pinl was overexpressed in melanoma, but it is not known whether Pin1 is involved in the oncogenesis of melanoma and it is unclear whether Pin1 inhibition would have effect on cellular growth or block tumorigenic properties.Therefore, In the present study, the expression of Pinl was silenced by miRNA interference in A375 cells to clarify whether Pinl suppression has effect on the abilities of proliferation and invasion of A375 in vitro and vivo.Firstly, we selected one miRNA plasmid with the most silence efficiency, miRNA plas4, from four miRNA plasmids provided by Invitrogen by western blot after transeint transfection. Then, by the method of stable transfection using miRNA plas4, we successfully established two clones, clone1 and clone2, with the silence efficiency of Pinl expression 84% and 85% respectively, compared with the vector control which we called mock.Secondly, in order to clarify whether Pinl is involved in the proliferation of MM, we evaluated the effects of Pinl depletion on A375 by cell proliferation assay and anchorage-dependent colony formation assay. As results showed, Pin1 inhibition resulted in a significant decrease in cell proliferation and colony formation. This indicated that Pin1 indeed played a positive role in promoting the proliferation of A375 cells. Furthermore, the Flow cytometry anaysis showed that depletion Pinl resulted in a directly arresting in G0/G1 phase.Thirdly, because MM is a serious skin cancer with abilities of high invasion and metastasis which lead to high mortality and poor prognosis of patients, in order to investigate whether Pin1 is associated with the invasion and metastasis of MM, we conducted the adhering assay, wound healing assay and transwell migration assay using A375 parental cells, mock and the two clones with Pin1 depletion. The results showed that suppression the Pin1 expression could significantly reduce the abilities of adhesion, invasion and metastasis of A375 cells.Moreover, in order to further explore the tumorigenic property of Pin1 in vivo, we monitored the tumor formation of Mock, Clone1 and Clone2 in nude mice. The results showed that inhibition of Pin1 could significantly reduce the tumorigenicity of A375 cells in nude mice.Finally, in order to elucidate whether these phenomenon were due to the inhibition of certain oncoproteins followed by Pin1 depletion, we tested the expression of some Pin1 related protein, already reported or not by western blot. The results showed that the activated Akt (showd as pAkt-Ser473/Akt), JNK and proMMP-2 which have major role in proliferation and invasion in MM were significantly reduced in Pin1 silenced clones and the expression of Her2 and CyclinD1 were down-regulated in clone2.In summary, Pin1 plays an important role in the proliferation and invasion of MM. Suppression Pin1 expression could inhibit a variety of oncoproteins at the same time to block tumorigenic properties and the development of cancer. Pin1 is likely to be a novel molecular target for MM therapy. Pinl, first identified in a yeast two-hybrid screen in 1996, is a protein interacting with the mitotic kinase NIMA(Never In Mitosis Gene A). Pinl is a member of parvulins which belongs to the peptidl-prolyl cis-trans isomerase (PPIase) family and can specifically recognize and isomerize the phosphotylated Ser/Thr-Pro bands in certain proteins, thereby changing their conformation and modulating their functions. A growing number of researches have been showed that Pinl is likely to be a novel anticancer target. Therefore, the study and development of Pinl inhibitors has become a hotspot in current research.There are mainly two kinds of Pinl inhibitors which is peptide inhibitor and small molecular inhibitor. Because peptide inhibitor has great difficulties in clinical application, the main direction of current research is small molecular inhibitor. Though after many years of efforts, more and more Pinl inhibitors with new structure have been discovered and some of which could bind to the PPIase with nanomolar affinity, for various reasons such as physical and chemical properties, until now, there still has been no specific and effective inhibitors of Pinl that could enter into the clinical trails. Therefore, it is essential to look for small molecular Pinl inhibitors with new structure.Based on the structure of Pinl enzyme and its substrate, the group of Professor Bailing Xu in Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College designed and synthesized a series of small molecular compounds with completely innovative structure combined with the molecular docking technology. We evaluated these compounds mainly by MTT screening and protease coupled PPIase assay and studied the mechanism of action preliminarily on one compound coded XP08075.First of all, we tested the Pin1 expression in 19 tumor cell lines from 10 different organ and tissues. We found that Pin1 is over expressed in cell lines of human prostate cancer, stomach cancer and cervical cancer whereas the human hepatoma carcinoma cell line HepG2 is the one with the lowest expression of Pinl in tested cell lines. Thus, we established a high expressing Pinl HepG2 cell line by stable transfection technology.Because MTT method can quickly evaluate the antitumor effects of compounds in vitro, we firstly screened 294 compounds by MTT assay. The results showed that there were 49 compounds with the IC50 of micromole level. Then, followed by rescreening of 9 compounds, we found that XP08075 had stronger inhibitory activity on cell proliferation and the sensitivity of tumor cells to XP08075 is correlated with the Pinl expression of their own. Thus, we further examined the antitumor activity of XP08075 on the established high expressing Pinl HepG2 cell model. Compared with the Mock which was transfected with vector, XP08075 exhibited more inhibitory effect on the high expressing Pinl HepG2 cell model.In order to elucidate whether these compounds with certain structure could inhibit the PPIase activity directly in vitro, we established the protease-coupled PPIase assay and detected the inhibitory effect of 51 compounds, including XP08075 on Pinl enzyme. Of these,9 compounds inhibit Pinl activity with IC50<10?M. In the case of relative good solubility, XLN418 is one of the most potent inhibitors with the IC50 of 4.9?M but the inhibitory effect of XP08075 is not good.Based on the analysis of the structure characteristics of effective compounds at molecular or cellular level, we found that most of the compounds with activity on cell level has eater as substituent whereas the compounds with activity on enzyme level always has carboxylic acid as substituent. The only structure difference between XLN418 and XP08075 is R3 substituent, which is formic in XLN418 and methyl ester in XP08075. Another two pairs of compounds also appeared the same pattern. Therefore, we speculate that after entering into the cells, the compound containing ester bonds may be partially hydrolyzed by esterase into a compound with carboxylic acid and then inhibit cell proliferation by depressing the Pinl activity. But because the cell membrane is not easily accessible for the compounds containing carboxylic acid, there is no inhibitory activity of these compounds at the cellular level. Based on the above speculation, we studied the antitumor mechanism of XP08075 preliminarily on MCF-7 cell line.The results of flow cytometry indicated that there were no significant effects on the cell cycle distribution of MCF-7 cells treated with 6?M XP08075 for 24,48 and 72 hours, but only a slight extension of G0/G1 phase of 72 hours exposure. The cell doubling time calculated from the growth curve showed that 6?M XP08075 could extend the doubling time of MCF-7 cells greatly, resulting in the extension of whole cell cycle progression.Then, we detected the protein expression of MCF-7 cells treated with XP08075 by western blot analysis. We observed that the expression of?-catenin and CyclinB 1 which regulated by Pinl indirectly was depressed in a time dependent manner. However, the expression of Pinl was also down regulated at the same time. Through the analysis of RT-PCR, we speculated that this kind of Pinl inhibition was largely originated form the increased degradation of Pinl.Therefore, it is likely that part of the XP08075 became to XLN418 by esterase followed by entering into the cells and played the antitumor effect by Pin1 inhibition.In summary, we believed that XLN418 could be a promising leading compound and lay a foundation in the study of Pin1 inhibitors with this kind of structure. XLN306, synthesized by professor Xu in our institute, is a noval compound with new structure which was mind to exhibit the antitumor activity by Pinl inhibition. Early results showed that although XLN306 could not significantly inhibit Pinl enzyme activity it could inhibit many kinds of tumor cell proliferation with the IC50 of micromole which the lowest one was human breast cancer cell line MX-1. Therefore, we choose MX-1 as a cell model in vitro to investigate the antitumor mechanism of XLN306.First of all, we detected the effects of cell cycle distribution and the apoptosis ratio of MX-1 treated with XLN306. The results showed that XLN306 could induce MX-1 cell apoptosis in dose-dependent and time-dependent manner. This indicated that XLN306 was likely to exhibit the antitumor effect by induction of apoptosis. In order to explore its mechanism of action, we have made a systematic and penetrating study focused on the different pathways and key points of apoptosis in vitro.By DAPI staining and DNA ladder experiments, we observed that XLN306 could induce MX-1 apoptosis with noticeable chromatin condensation and nuclear fragmentation phenomenon and typical DNA ladder resulted in nucleosome DNA breakage.The results of western blot analysis indicated that XLN306 could activate the death receptor pathway and mitochondrial pathway, the two classic pathways of apoptosis at the same time, by increasing the expression of Fas and FasL, reducing the ratio of Bcl2/Bax and promoting the release of Cytochrome C, which together further activate Caspase3 and lead to apoptosis finally. In addition,20?mol/L XLN306 could decrease the phosphorylation of Akt significantly which play important role in survival pathway and promote apoptosis by down-regulating the activity of Akt and up-regulating the activity of GSK3?.In order to explore the antitumor activity of XLN306 in vivo, we carried out the experimental study on XLN306 in the treatment of H22 hepatocarcinoma transplanted in mice. The results showed that XLN306 could inhibit tumor growth significantly and had a better dose-activity relationship.In summary, the results in vitro and in vivo showed that XLN306 could inhibit tumor proliferation by inducing apoptosis through death receptor and mitochondrial pathways.