Novel Molecular Targets of Azadirachta indica Associated with Inhibition of Tumor Growth in Prostate Cancer

Advanced prostate cancer has significant long-term morbidity, and there is a growing interest in alternative and complimentary forms of therapy that will improve the outcomes of patients. Azadirachta indica (common name: neem) contains multiple active compounds that have potent anti-inflammatory and anticancer properties. The present study investigates the novel targets of the anticancer activity of ethanol extract of neem leaves (EENL) in vitro and evaluates the in vivo efficacy in the prostate cancer models. Analysis of the components in the EENL by mass spectrometry suggests the presence of 2′,3′-dehydrosalannol, 6-desacetyl nimbinene, and nimolinone. Treatment of C4-2B and PC-3M-luc2 prostate cancer cells with EENL inhibited the cell proliferation. Genome-wide expression profiling, using oligonucleotide microarrays, revealed genes differentially expressed with EENL treatment in prostate cancer cells. Functional analysis unveiled that most of the up-regulated genes were associated with cell death, and drug metabolism, and the down-regulated genes were associated with cell cycle, DNA replication, recombination, and repair functions. Quantitative PCR confirmed significant up-regulation of 40 genes and immunoblotting revealed increase in the protein expression levels of HMOX1, AKR1C2, AKR1C3, and AKR1B10. EENL treatment inhibited the growth of C4-2B and PC-3M-luc2 prostate cancer xenografts in nude mice. The suppression of tumor growth is associated with the formation of hyalinized fibrous tumor tissue and the induction of cell death by apoptosis. These results suggest that EENL-containing natural bioactive compounds could have potent anticancer property and the regulation of multiple cellular pathways could exert pleiotrophic effects in prevention and treatment of prostate cancer

Differential Gene Expression and Adherence of Escherichia coli O157:H7 In Vitro and in Ligated Pig Intestines

BACKGROUND: Escherichia coli O157:H7 strain 86-24 grown in MacConkey broth (MB) shows almost no adherence to cultured epithelial cells but adheres well in pig ligated intestines. This study investigated the mechanisms associated with the difference between in-vitro and in-vivo adherence of the MB culture. METHODOLOGY/PRINCIPAL FINDINGS: It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR). Expression of selected virulence-related genes associated with adherence and CCR was then examined by quantitative PCR. When bacteria were grown in MB and Brain Heart Infusion with NaHCO(3) (BHIN) plus lactose, pH was reduced to 5.5-5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE) genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP), and two negative regulators of the LEE, gadE and hfq. Putative virulence genes stcE, hlyA, ent and nleA were also decreased in vitro. Reversal of these changes was noted for bacteria recovered from the intestine, where transcripts for qseF and fis and putative virulence factors AidA(15), TerC and Ent/EspL2 were significantly increased, and transcripts for AIDA(48), Iha, UreC, Efa1A, Efa1B, ToxB, EhxA, StcE, NleA and NleB were expressed at high levels. CONCLUSIONS/SIGNIFICANCE: Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo. CCR and/or acidic pH may have played a role in repression of the LEE genes. Bacterial pathogens need to integrate their nutritional metabolism with expression of virulence genes but little is known of how this is done in E. coli O157:H7. This study indicates one aspect of the subject that should be investigated further

Modulation of host cell processes by T3SS effectors

Two of the enteric Escherichia coli pathotypes-enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC)-have a conserved type 3 secretion system which is essential for virulence. The T3SS is used to translocate between 25 and 50 bacterial proteins directly into the host cytosol where they manipulate a variety of host cell processes to establish a successful infection. In this chapter, we discuss effectors from EPEC/EHEC in the context of the host proteins and processes that they target-the actin cytoskeleton, small guanosine triphosphatases and innate immune signalling pathways that regulate inflammation and cell death. Many of these translocated proteins have been extensively characterised, which has helped obtain insights into the mechanisms of pathogenesis of these bacteria and also understand the host pathways they target in more detail. With increasing knowledge of the positive and negative regulation of host signalling pathways by different effectors, a future challenge is to investigate how the specific effector repertoire of each strain cooperates over the course of an infection

Microarray analysis of the Ler regulon in enteropathogenic and enterohaemorrhagic Escherichia coli strains

The type III protein secretion system is an important pathogenicity factor of enteropathogenic and enterohaemorrhagic Escherichia coli pathotypes. The genes encoding this apparatus are located on a pathogenicity island (the locus of enterocyte effacement) and are transcriptionally activated by the master regulator Ler. In each pathotype Ler is also known to regulate genes located elsewhere on the chromosome, but the full extent of the Ler regulon is unclear, especially for enteropathogenic E. coli. The Ler regulon was defined for two strains of E. coli: E2348/69 (enteropathogenic) and EDL933 (enterohaemorrhagic) in mid and late log phases of growth by DNA microarray analysis of the transcriptomes of wild-type and ler mutant versions of each strain. In both strains the Ler regulon is focused on the locus of enterocyte effacement – all major transcriptional units of which are activated by Ler, with the sole exception of the LEE1 operon during mid-log phase growth in E2348/69. However, the Ler regulon does extend more widely and also includes unlinked pathogenicity genes: in E2348/69 more than 50 genes outside of this locus were regulated, including a number of known or potential pathogenicity determinants; in EDL933 only 4 extra-LEE genes, again including known pathogenicity factors, were activated. In E2348/69, where the Ler regulon is clearly growth phase dependent, a number of genes including the plasmid-encoded regulator operon perABC, were found to be negatively regulated by Ler. Negative regulation by Ler of PerC, itself a positive regulator of the ler promoter, suggests a negative feedback loop involving these proteins