新型p-21活化激酶4抑制剂的研发进展

目的研究p-21活化激酶4(p21-activated kinase 4,PAK4)抑制剂在肿瘤治疗中的应用,为新型PAK4抑制剂的研发提供参考。方法从母核结构、共晶结构、药物活性、药代动力学、作用机制等方面,对小分子PAK4抑制剂进行论述。结果在多种PAK4抑制剂结构类型中,苯并呋喃类化合物KPT-9274对PAK4的抑制活性显著,对多种癌症具有抗肿瘤作用,是目前唯一处于临床试验阶段的PAK4抑制剂,可以作为今后PAK4抑制剂设计的基础。结论对苯并呋喃类化合物进一步结构优化,有望获得活性更优的PAK4抑制剂用于抗肿瘤研究。

Group Ⅱ p21-activated kinases as therapeutic targets in gastrointestinal cancer

P21-activated kinases(PAKs) are central players in various oncogenic signaling pathways. The six PAK family members are classified into group Ⅰ(PAK1-3) and group Ⅱ(PAK4-6). Focus is currently shifting from group Ⅰ PAKs to group Ⅱ PAKs. Group Ⅱ PAKs play important roles in many fundamental cellular processes, some of which have particular significance in the development and progression of cancer. Because of their important functions, group Ⅱ PAKs have become popular potential drug target candidates. However, few group Ⅱ PAKs inhibitors have been reported, and most do not exhibit satisfactory kinase selectivity and "drug-like" properties. Isoform- and kinase-selective PAK inhibitors remain to be developed. This review describes the biological activities of group Ⅱ PAKs, the importance of group Ⅱ PAKs in the development and progression of gastrointestinal cancer, and smallmolecule inhibitors of group Ⅱ PAKs for the treatment of cancer.

Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.

p21 and p27 immunoexpression in gastric well differentiated endocrine tumors(ECL-cell carcinoids)

AIM： To investigate the expression of cyclin-dependent kinase inhibitors p21 and p27 in gastric well differentiated endocrine tumors （GWDET） （ECLocell carcinoids）.METHODS： The expressions of p21 and p27 were examined immunhistochemically in endoscopic biopsy specimens from 16 patients matching the diagnostic criteria of GWDET. Percentage of positive nuclear staining either weak or strong was noted. The association of immunoexpressions with age, gender, tumor localization, multifocality and accompanying chronic atrophic gastritis, neuroendocrine cell hyperplasia （NEH）, neuroendocrine dysplasia （NED）, intestinal metaplasia （IM）, Ki-67 proliferation index and clinical outcome were also evaluated.RESULTS： All cases expressed p27 with a mean expression score of 43.6%, while 31.3% of the cases showed any p21 expression, p21 and p27 immunoexpressions were significantly correlated with each other （P 〈 0.01）, and the p21-expressing group had higher p27 expression scores （68% vs 22%）. p21 and p27 expressions were lower in women, in non-atrophic mucosa and cases whose tumors were located somewhere other than fundus without submucosal extension. On contrary, p21 and p27 expressions were higher in males and the patients with submucosal extension and atrophic gastritis. Cases presenting lower p27 scores had solitary tumors showing neither NEH-NED nor IM. Despite, cases with lower p21 expression presented multifocal tumors accompanied by NEH-NED. However, no correlation of p21 and p27 expressions was found with age and Ki-67 expression.CONCLUSION： p27 is widely expressed in GWDETs, while p21 expression is sparse and observed in two thirds of the cases. Loss of p21 and p27 expressions may be correlated with different carcinoid tumor subtypes; however,more studies are needed to assess the role of these prospective markers in gastrointestinal endocrine tumors.

Targeting P21-activated kinase suppresses proliferation and enhances chemosensitivity in T-cell lymphoblastic lymphoma

T-cell lymphoblastic lymphoma(T-LBL)is a highly aggressive non-Hodgkin lymphoma with a poor prognosis.P21-activated kinase(PAK)is a component of the gene expression-based classifier that can predict the prognosis of T-LBL.However,the role of PAK in T-LBL progression and survival remains poorly understood.Herein,we found that the expression of PAK1 was significantly higher in T-LBL cell lines(Jurkat,SUP-T1,and CCRF-CEM)compared to the human T-lymphoid cell line.Moreover,PAK2 mRNA level of 32 relapsed T-LBL patients was significantly higher than that of 37 cases without relapse(P=.012).T-LBL patients with high PAK1 and PAK2 expression had significantly shorter median RFS than those with low PAK1 and PAK2 expression(PAK1,P=.028;PAK2,P=.027;PAK1/2,P=.032).PAK inhibitors,PF3758309(PF)and FRAX597,could suppress the proliferation of T-LBL cells by blocking the G1/S cell cycle phase transition.Besides,PF could enhance the chemosensitivity to doxorubicin in vitro and in vivo.Mechanistically,through western blotting and RNA sequencing,we identified that PF could inhibit the phosphorylation of PAK1/2 and downregulate the expression of cyclin D1,NF-κB and cell adhesion signaling pathways in T-LBL cell lines.These findings suggest that PAK might be associated with T-LBL recurrence and further found that PAK inhibitors could suppress proliferation and enhance chemosensitivity of T-LBL cells treated with doxorubicin.Collectively,our present study underscores the potential therapeutic effect of inhibiting PAK in T-LBL therapy.

Molecular determinants of the antitumor effects of trichostatin A in pancreatic cancer cells

AIM:To gain molecular insights into the action of the histone deacetylase inhibitor(HDACI) trichostatin-A(TSA) in pancreatic cancer(PC) cells.METHODS:Three PC cell lines,BxPC-3,AsPC-1 and CAPAN-1,were treated with various concentrations of TSA for def ined periods of time.DNA synthesis was assessed by measuring the incorporation of 5-bromo-2'deoxyuridine.Gene expression at the level of mRNA was quantif ied by real-time polymerase chain reaction.Expression and phosphorylation of proteins was monitored by immunoblotting,applying an infrared imaging technology.To study the role of p38 MAP kinase,the specif ic enzyme inhibitor SB202190 and an inactive control substance,SB202474,were employed.RESULTS:TSA most eff iciently inhibited BrdU incorporation in BxPC-3 cells,while CAPAN-1 cells displayed the lowest and AsPC-1 cells an intermediate sensitivity.The biological response of the cell lines correlated with the increase of histone H3 acetylation after TSA application.In BxPC-3 cells(which are wild-type for KRAS),TSA strongly inhibited phosphorylation of ERK 1/2 and AKT.In contrast,activities of ERK and AKT in AsPC-1 and CAPAN-1 cells(both expressing oncogenic KRAS) were not or were only modestly affected by TSA treatment.In all three cell lines,but most pronounced in BxPC-3 cells,TSA exposure induced an activation of the MAP kinase p38.Inhibition of p38 by SB202190 slightly but signif icantly diminished the antiproliferative effect of TSA in BxPC-3 cells.Interestingly,only BxPC-3 cells responded to TSA treatment by a signif icant increase of the mRNA levels of bax,a pro-apoptotic member of the BCL gene family.Finally,in BxPC-3 and AsPC-1 cells,but not in the cell line CAPAN-1,signif icantly higher levels of the cell cycle inhibitor protein p21Waf1 were observed after TSA application.CONCLUSION:The biological effect of TSA in PC cells correlates with the increase of acetyl-H3,p21Waf1,phospho-p38 and bax levels,and the decrease of phosphoERK 1/2 and phospho-AKT.

Effect of cis-9,trans-11-conjugated linoleic acid on cell cycle of gastric adenocarcinoma cell line(SGC-7901)

AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B(1), D(1), p16(ink4a) and p21(cip/waf1) of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25, 50, 100 and 200 micromol.L(-1))of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane). RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c9, t11-CLA.SGC-7901 cells. Eight day after treatment with various concentrations of c9, t11-CLA mentioned above, the inhibition rates were 5.92%, 20.15%, 75.61% and 82.44%, respectively and inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 micromol.L, 24h) showed significantly less (3)H-TdR incorporation than that in the negative controls (P【0.05 and P【0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%, 24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B(1) (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D(1) (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B(1) and D(1) were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P【0.01), whereas the expressions of P16(ink4a) and P21(cip/waf1), cyclin-dependent kinases inhibitors(CDKI), were increased. CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c9, t11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B(1) and D(1) and enhanced expressions of CDKI(P16(ink4a) and p21(cip/waf1)).

Effect of Yanggan Jiedu Sanjie formula on human hepatocellular carcinoma Bel-7402 cells

OBJECTIVE: To observe the effect of Yanggan Jiedu Sanjie(YGJDSJ) formula on human hepatocellular carcinoma Bel-7402 cells.METHODS: Bel-7402 cells were treated with YGJDSJ. Cell proliferation was detected by cell counting kit-8 assay. Cell apoptosis was identified by Hoechst 33258 staining and flow cytometric analysis. Cell cycle distribution was quantified by flow cytometric analysis. Caspase activities were measured by commercial kit. Cell senescence was detected by senescence-associated β-galactosidase(SA-β-gal)staining. Protein expression and phosphorylation were identified by Western blot. Protein expression was knocked-down by siRNA.RESULTS: YGJDSJ inhibited proliferation of Bel-7402 cells in a dose-and time-dependent manner.YGJDSJ induced apoptosis and activated caspase-3, 8, and 9 in Bel-7402 cells. YGJDSJ-induced apoptosis was completely abrogated by a pan caspase inhibitor, Z-VAD-FMK. YGJDSJ also induced cell senescence, up-regulated cyclin-dependent kinase inhibitor 1 a(CDKN1 a) and CDKN2 a expression and down-regulated retinoblastoma protein(RB) phosphorylation in Bel-7402 cells. Specific knockdown of CDKN1 a and CDKN2 a significantly reduced YGJDSJ-induce cell senescence in Bel-7402 cells.CONCLUSION: YGJDSJ inhibited cell proliferation,induced caspase-dependent apoptosis and CDKN1 a/CDKN2 a-RB signalling mediated cell senescence in Bel-7402 cells. Our findings suggest that YGJDSJ might be potential for hepatocellular carcinoma treatment.