Inhibition of EGFR-AKT Axis Results in the Suppression of Ovarian Tumors In Vitro and in Preclinical Mouse Model

Ovarian cancer is the leading cause of cancer related deaths in women. Genetic alterations including overexpression of EGFR play a crucial role in ovarian carcinogenesis. Here we evaluated the effect of phenethyl isothiocyanate (PEITC) in ovarian tumor cells in vitro and in vivo. Oral administration of 12 μmol PEITC resulted in drastically suppressing ovarian tumor growth in a preclinical mouse model. Our in vitro studies demonstrated that PEITC suppress the growth of SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells by inducing apoptosis in a concentration-dependent manner. Growth inhibitory effects of PEITC were mediated by inhibition of EGFR and AKT, which are known to be overexpressed in ovarian tumors. PEITC treatment caused significant down regulation of constitutive protein levels as well as phosphorylation of EGFR at Tyr1068 in various ovarian cancer cells. In addition, PEITC treatment drastically reduced the phosphorylation of AKT which is downstream to EGFR and disrupted mTOR signaling. PEITC treatment also inhibited the kinase activity of AKT as observed by the down regulation of p-GSK in OVCAR-3 and TOV-21G cells. AKT overexpression or TGF treatment blocked PEITC induced apoptosis in ovarian cancer cells. These results suggest that PEITC targets EGFR/AKT pathway in our model. In conclusion, our study suggests that PEITC could be used alone or in combination with other therapeutic agents to treat ovarian cancer.     

热带地区相对湿度的季节性变化影响果蝇Drosophila jambulina体色多型性和水分平衡(英文)

在印度次大陆的亚热带地区, 秋天冷而干燥, 春天湿润。变温性果蝇所具有的抗干燥性有助于其度过较为干旱的气候条件。 Drosophila jambulina 具有体色二型性。已有研究表明, 随湿度变化, D. jambulina热带种群始终保持体色多型性, 这与热条件下体色黑化相反, 且该热带物种中体色分化频率随季节性变化, 这符合黑化 干燥假说。但是两种色型的D. jambulina产生这类气候适应的机理尚不明了。为了检验干燥相关性状生理基础的分化与对气候条件的色型特异性适应相关这一假说, 我们利用分别在17℃和25℃、 低湿（40% RH）和高湿（80% RH）条件下饲养获得的两种色型的D. jambulina, 检测了其水分平衡对相对湿度、 温度、 及温湿度相互作用的反应。我们发现, 在低相对湿度下, 两种温度下饲养的深色型果蝇的生理和脱水性状数值显著高于浅色型。对两种色型果蝇的水分收支情况进行的比较分析表明, 在低相对湿度下, 深色型果蝇的含水量较高、 水分损失率较低、 抗脱水能力较强, 使其具有更强的抗干燥性。在干燥胁迫过程中, 两种色型的果蝇均以碳水化合物作为代谢燃料, 但是在低湿条件下, 深色果蝇中贮存碳水化合物的含量明显要高。而且, 在两种湿度条件下, 这两种色型果蝇之间的总能量收支显著不同。据此认为, D. jambulina的水分平衡相关性状表现出的色型特异性分化与其对湿热生境的适应相关。     

Analysis of chromatin structure of genes silenced by heterochromatin in trans

While heterochromatic gene silencing in cis is often accompanied by nucleosomal compaction, characteristic histone modifications, and recruitment of heterochromatin proteins, little is known concerning genes silenced by heterochromatin in trans. An insertion of heterochromatic satellite DNA in the euchromatic brown (bw) gene of Drosophila melanogaster results in bwDominant (bwD), which can inactivate loci on the homolog by relocation near the centric heterochromatin (trans-inactivation). Nucleosomal compaction was found to accompany trans-inactivation, but stereotypical heterochromatic histone modifications were mostly absent on silenced reporter genes. HP1 was enriched on trans-inactivated reporter constructs and this enrichment was more pronounced on adult chromatin than on larval chromatin. Interestingly, this HP1 enrichment in trans was unaccompanied by an increase in the 2MeH3K9 mark, which is generally thought to be the docking site for HP1 in heterochromatin. However, a substantial increase in the 2MeH3K9 mark was found on or near the bwD satellite insertion in cis, but did not spread further. These observations suggest that the interaction of HP1 with chromatin in cis is fundamentally different from that in trans. Our molecular data agree well with the differential phenotypic effect on bwD trans-inactivation of various genes known to be involved in histone modification and cis gene silencing.     

Nicotinamide inhibits Plasmodium falciparum Sir2 activity in vitro and parasite growth

Plasmodium falciparum sirtuin, PfSir2, contains histone deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs.     

Plasmodium falciparum origin recognition complex subunit 5: functional characterization and role in DNA replication foci formation

The mechanism of DNA replication initiation and progression is poorly understood in the parasites, including human malaria parasite Plasmodium falciparum . Using bioinformatics tools and yeast complementation assay, we identified a putative homologue of Saccharomyces cerevisiae o rigin r ecognition c omplex subunit 5 in P. falciparum (PfORC5). PfORC5 forms distinct nuclear foci colocalized with the replication foci marker proliferating cell nuclear antigen (PfPCNA) and co-immunoprecipitates with PCNA during early-to-mid trophozoite stage replicating parasites. Interestingly, these proteins separate from each other at the non-replicating late schizont stage, citing the evidence of the presence of both PCNA and ORC components in replication foci during eukaryotic DNA replication. PfORC1, another ORC subunit, colocalizes with PfPCNA and PfORC5 at the beginning of DNA replication, but gets degraded at the late schizont stage, ensuring the regulation of DNA replication in the parasites. Further, we have identified putative PCNA-interacting protein box in PfORC1 that may explain in part the colocalization of PfORC and PfPCNA. Additionally, use of specific DNA replication inhibitor hydroxyurea affects ORC5/PCNA foci formation and parasitic growth. These results strongly favour replication factory model in the parasites and confer great potential to understand the co-ordination between ORC and PCNA during eukaryotic DNA replication in general.     

Differentiation Induces Dramatic Changes in miRNA Profile,Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.