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Biomedical subjects

Saghir Akhtar

Publications and source records attributed to Saghir Akhtar.

24 records · Page 2Linked to original sources
PubMed2003

Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system.

Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC50 values of around 750 nM. In contrast, siRNA was significantly more potent with an IC50 of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.

Blotting, Western↗
PubMed2002

Sequence and chemistry requirements for a novel aptameric oligonucleotide inhibitor of EGF receptor tyrosine kinase activity.

We have previously identified a phosphorothioate oligonucleotide (PS-ODN) that inhibited epidermal growth factor receptor tyrosine kinase (TK) activity both in cell fractions and in intact A431 cells. Since ODN-based TK inhibitors may have anti-cancer applications and may also help understand the non-antisense mediated effects of PS-ODNs, we have further studied the sequence and chemistry requirements of the parent PS-ODN (sequence: 5'-GGA GGG TCG CAT CGC-3') as a sequence-dependent TK inhibitor. Sequence deletion and substitution studies revealed that the 5'-terminal GGA GGG hexamer sequence in the parent compound was essential for anti-TK activity in A431 cells. Site-specific substitution of any G with a T in this 5'-terminal motif within the parent compound caused a significant loss in anti-TK activity. The fully PS-modified hexameric motif alone exhibited equipotent activity as the parent 15-mer whereas phosphodiester (PO) or 2'-O-methyl-modified versions of this motif had significantly reduced anti-TK activity. Further, T substitutions within the two 5'-terminal G residues of the hexameric PS-ODN to produce a sequence, TTA GGG, representing the telomeric repeats in human chromosomes, also did not exhibit a significant anti-TK activity. Multiple repeats of the active hexameric motif in PS-ODNs resulted in more potent inhibitors of TK activity than the parent ODN. These results suggested that PS-ODNs, but not PO or 2'-O-methyl modified ODNs, containing the GGA GGG motif can exert potent anti-TK activity which may be desirable in some anti-tumor applications. Additionally, the presence of this previously unidentified motif in antisense PS-ODN constructs may contribute to their biological effects in vitro and in vivo and should be accounted for in the design of the PS-modified antisense ODNs.

Animals↗
PubMed2002

Co-delivery of an antisense oligonucleotide and 5-fluorouracil using sustained release poly (lactide-co-glycolide) microsphere formulations for potential combination therapy in cancer.

Antisense oligonucleotides (AODNs) can selectively inhibit oncogene expression by Watson-Crick hybridisation to target mRNA and are being increasingly considered for use in combination with conventional drugs for potential anticancer therapy. Combination therapy of AODNs and cytotoxic agents using biodegradable polymeric delivery systems potentially offers several advantages including site-specific or organ-directed targeting, protection from digesting enzymes, and improved pharmacokinetics/pharmacodynamics resulting from sustained delivery of the entrapped drugs. Using a model AODN targeting the epidermal growth factor receptor (that is over-expressed in several cancers including breast and brain cancer) and the commonly used cytotoxic agent, 5-fluorouracil (5-FU), we have examined the use of poly (lactide-co-glycolide) (P(LA-GA)) microsphere formulations for co-delivery of these agents. Both agents were either co-entrapped in a single microsphere formulation or individually entrapped in two separate microsphere formulations and release profiles determined in vitro. Using a double emulsion method for preparing the P(LA-GA) microspheres suitable entrapment and sustained release over 35 days was observed in both types of formulation. Release of AODN and 5-FU from all formulations appeared to be biphasic. However, the release rates of the two agents were significantly slower when co-entrapped as a single microsphere formulation compared to those obtained with the separate formulations. Electrophoretic mobility shift assays suggested that this might be, in part, due to an interaction of 5-FU with the oligodeoxynucleotide (ODN). Further, our data suggest that by mixing individual formulations of 5-FU and ODNs at different mass ratios allowed greater flexibility in achieving the desired release profile as well as avoiding potential drug-drug interactions. Thus, co-administration of individual P(LA-GA) microsphere formulations of AODNs and 5-FU, at appropriate mass ratios, appears worthy of further investigation for the potential co-delivery of these anti-cancer agents in vivo.

Antimetabolites, Antineoplastic↗
PubMedDate not supplied

Phosphoinositide 3-kinase mediated signalling contributes to development of diabetes-induced abnormal vascular reactivity of rat carotid artery.

Diabetes mellitus is associated with vascular complications, including an impairment of vascular function and alterations in the reactivity of blood vessels to vasoactive agents. Phosphatidylinositol 3-kinase (PI3K) is a signalling enzyme that plays key roles in vascular growth, proliferation and cellular apoptosis and is implicated in modulating vascular smooth muscle contractility. The aim of this study was to determine whether PI3K plays a role in development of diabetes-induced altered vascular reactivity to selected vasoconstrictors and vasodilators. The effect of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a selective PI3K inhibitor, on isolated segments of carotid arteries from streptozotocin (STZ)-diabetic rats was investigated. Ring segments of the isolated carotid arteries were mounted in organ baths to measure changes in isometric tension. Our results showed that STZ treatment produced an increase in the vasoconstrictor response to norepinephrine (NE), angiotensin II (Ang II) and endothelin-1 (ET-1) and an attenuated vasodilator response to carbachol and histamine in the isolated carotid arteries from STZ-diabetic animals. Diabetes-induced impaired vascular responsiveness to the vasoactive agonists was prevented by chronic inhibition of PI3K by LY294002 even though blood glucose levels remained high. This is the first study to show that selective inhibition of PI3K can attenuate the development of diabetes-induced abnormal vascular reactivity in the isolated carotid arteries of diabetic rats.

1-Phosphatidylinositol 4-Kinase↗
PubMedDate not supplied

Inhibition of Ras-GTPase improves diabetes-induced abnormal vascular reactivity in the rat perfused mesenteric vascular bed.

OBJECTIVE: The signalling mechanisms involved in regulating altered vascular reactivity in diabetes are not fully understood. The aim of this study was to investigate the role of Ras-GTPase in the development of abnormal vascular reactivity in diabetes. MATERIALS AND METHODS: We investigated the ability of chronic administration of FPTIII (1.5 mg/kg), an inhibitor of Ras-GTPase, to modulate the altered vasoreactivity of the rat perfused mesenteric bed to common vasoconstrictors and vasodilators in streptozotocin (STZ)-induced diabetes. RESULTS: The vasoconstrictor responses induced by norepinephrine (NE) and endothelin-1 (ET-1) were significantly increased whereas vasodilator responses to carbachol, histamine and isoprenaline were significantly reduced in the perfused mesenteric bed of the STZ-diabetic rats. Inhibition of Ras-GTPase by chronic administration of FPTIII produced a significant normalization of the altered agonist-induced vasoconstrictor and vasodilator responses without affecting blood glucose levels. Inhibition of Ras-GTPase did not affect the agonist-induced vasoconstrictor and vasodilator responses in the control animals. CONCLUSION: These data suggest that signal transduction pathways activated by Ras-GTPase are involved in the development of diabetic vascular dysfunction. Potential strategies aimed at modifying actions of signal transduction pathways involving Ras-GTPase may therefore prove to be beneficial in treatment of vascular complications in diabetes.

Animals↗