SPARC promoter hypermethylation in colorectal cancers can be reversed by 5-Aza-2′deoxycytidine to increase SPARC expression and improve therapy response

Poor clinical outcomes in cancer can often be attributed to inadequate response to chemotherapy. Strategies to overcome either primary or acquired chemoresistance may ultimately impact on patients' survival favourably. We previously showed that lower levels of SPARC were associated with therapy-refractory colorectal cancers (CRC), and that upregulating its expression enhances chemo-sensitivity resulting in greater tumour regression in vivo. Here, we examined aberrant hypermethylation of the SPARC promoter as a potential mechanism for repressing SPARC in CRCs and whether restoration of its expression with a demethylating agent 5-Aza-2′deoxycytidine (5-Aza) could enhance chemosensitivity. Initially, the methylation status of the SPARC promoter from primary human CRCs were assessed following isolation of genomic DNA from laser capture microdissected specimens by direct DNA sequencing. MIP101, RKO, HCT 116, and HT-29 CRC cell lines were also used to evaluate the effect of 5-Aza on: SPARC promoter methylation, SPARC expression, the interaction between DNMT1 and the SPARC promoter (ChIP assay), cell viability, apoptosis, and cell proliferation. Our results revealed global hypermethylation of the SPARC promoter in CRCs, and identified specific CpG sites that were consistently methylated in CRCs but not in normal colon. We also demonstrate that SPARC repression in CRC cell lines could be reversed following exposure to 5-Aza, which resulted in increased SPARC expression, leading to a significant reduction in cell viability (by an additional 39% in RKO cells) and greater apoptosis (an additional 18% in RKO cells), when combined with 5-FU in vitro (in comparison to 5-FU alone). Our exciting findings suggest potential diagnostic markers of CRCs based on specific methylated CpG sites. Moreover, the results reveal the therapeutic utility of employing demethylating agents to improve response through augmentation of SPARC expression

Synthesis of three branched iminosugars [(3R,4R,5S)-3-(hydroxymethyl)piperidine-3,4,5-triol, (3R,4R,5R)-3-(hydroxymethyl)piperidine-3,4,5-triol and (3S,4R,5R)-3-(hydroxymethyl)piperidine-3.4,5-triol] and a branched trihydroxynipecotic acid [(3R,4R,5R)-3,4,5-trihydroxypiperidine-3-carboxylic acid] from sugar lactones with a carbon substituent at C-2

Homochiral piperidines containing a quaternary carbon branch at C-2 of the piperidine ring may be made from readily available carbohydrate lactones containing a branched 2-C-hydroxymethyl substituent. This gives an easy access to the branched iminosugars. In particular a trihydroxypipecolic acid analogue, (3R,4R,5R)-3,4,5-trihydroxypiperidine-3-carboxylic acid, has been synthesised efficiently from the starting material d-ribose and (3S,4R,5S)-3-(hydroxymethyl) piperidine-3,4,5-triol was found to be a specific inhibitor of α-d-glucosidase from Bacillus Stearothermophilus with an IC 50 value of 93 μM. © 2012 Elsevier Ltd. All rights reserved

Hydroxymethyl-Branched Piperidines from Hydroxymethyl-Branched Lactones: Synthesis and Biological Evaluation of 1,5-Dideoxy-2-C-hydroxymethyl-1,5-imino-D-mannitol, 1,5-Dideoxy-2-C-hydroxymethyl-1,5-imino-L-gulitol and 1,5-Dideoxy-2-C-hydroxymethyl-1,5-imino-D-talitol

Three homochiral polyhydroxylated piperidines containing a quaternary carbon branch at C-2 of the heterocyclic ring, which can be considered as branched analogues of deoxymannorijimycin (DMJ), and their corresponding lactams were synthesised from readily available and versatile carbohydrate lactones containing a 2-C-hydroxymethyl branch. The key step in the synthesis of these iminosugars, 1,5-dideoxy-2-C-hydroxymethyl-1,5-imino-D-mannitol, 1,5-dideoxy-2-C-hydroxymethyl-1,5-imino-L-gulitol and 1,5-dideoxy-2-C- hydroxymethyl-1,5-imino-D-talitol, was an efficient modified Kiliani reaction performed on the readily available ketose starting materials L-sorbose and D-fructose. The introduction of the carbon branch at C-2 resulted in loss of glycosidase inhibitory activity, which could be advantageous for therapeutic use of such compounds as chaperones. Here we report the preparation of three branched iminosugars, which can be considered as analogues of deoxymannorijimicin (DMJ) containing a quaternary carbon branch at C-2, and their corresponding lactams. The starting materials are branched sugar lactone intermediates, which in turn were afforded by a modified Kiliani procedure from L-sorbose and D-fructose. © 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim

Studies on the transformation of nitrosugars into branched chain iminosugars. Part II: Synthesis of (3R,4R,5R,6S)-2,2-bis(hydroxymethyl)azepane-3,4,5,6-tetraol

The first stereocontrolled synthesis of (3R,4R,5R,6S)-2,2-bis-(hydroxymethyl)azepane-3,4,5,6-tetraol is described herein. The method involves a novel double Henry reaction of the 3,5-di-O-benzyl-6-deoxy-1,2-O-isopropylidene-6-nitro-α-d-glucofuranose with formaldehyde followed by a reductive ring closure to give the first branched 1,6-dideoxy-1,6-heptitol described. © 2008 Elsevier Ltd. All rights reserved

Preliminary studies on the transformation of nitrosugars into branched chain iminosugars: synthesis of 1,4-dideoxy-4-C-hydroxymethyl-1,4-imino-pentanols.

A novel promising strategy for the transformation of nitrosugars into branched pyrrolidines, based on double Henry reaction with formaldehyde followed by reductive ring closure, allowed the first enantiospecific synthesis of a 4-C-hydroxymethyl branched derivative of the well-known glycosidase inhibitor 1,4-dideoxy-1,4-imino-pentanol. This strategy also afforded a new route to some other interesting derivatives, such as N-hydroxy, N-propyloxy, and imino derivatives, a new kind of compounds with promising biological properties. [reaction: see text]