With the PSH-L, the levels of PFKFB3 mRNA gradually decreased with increasing concentration of pshPFKFB3 up to 40 ng level

With the PSH-L, the levels of PFKFB3 mRNA gradually decreased with increasing concentration of pshPFKFB3 up to 40 ng level. diminution of pGP activity leading to changes in cell cycle (Cdk2), survival (survivin), apoptosis (Bcl2 and cleaved caspase 3) and stress (p-JNK and p-p38) markers so that induces apoptosis by PSH-DL in NSCLC cells. PSH-DL also showed ~3.8-fold reduction in tumor volume in A549 xenograft model which was significantly higher than individual treatments alone. Conclusion Targeting PFKFB3 through shRNA based RNAi is a promising approach for potentiating activity of DTX in NSCLC. stability and therapeutic potential (14). Docetaxel (DTX) has been used as a primary agent for the treatment Trilaciclib of solid tumors such as in lung, breast and pancreatic cancers (15C17). Currently available marketed formulations of taxanes (DTX and paclitaxel) face issues of ZNF143 resistance development and toxicities which are dose limiting (18). Abraxane, which is a nanoparticle albumin-bound paclitaxel, has been Trilaciclib approved for metastatic pancreatic, lung and breast cancer. It is used in combination with carboplatin for the initial treatment of patients with locally advanced or metastatic NSCLC who are not candidates for curative surgery or radiation therapy (19). Taxotere and taxol, which are micellar formulations of DTX and paclitaxel, respectively, are used for intravenous therapy of breast, lung, prostate, gastric, head and neck, ovarian and pancreatic cancer. However, both the drugs have dose dependent side effects like neutropenia, alopecia and anemia. Hence, a formulation strategy is required that can overcome its toxicity while providing better alternative treatment approach in comparison to those available in the clinic. Liposomes are able to entrap hydrophobic drugs in the bilayer and provide protection from degradation, reduce toxicity, and possibly help in overcoming multidrug resistance (20). Furthermore, liposomes can be modified using appropriate selection of lipids to load large hydrophobic drugs such Trilaciclib as DTX in the bilayer along with the use of cationic lipids to complex with therapeutic genes (21). Liposomal co-delivery of the drug with the shRNA plasmid against PFKFB3 would be of added advantage by providing a simultaneous delivery of drug and gene using the same nano-particulate carrier to the tumor site. The objective of this study was to evaluate the therapeutic potential of plasmids to provide pool of shRNA in cells for silencing PFKFB3 and evaluate the efficacy of the co-delivery of a chemotherapeutic agent with such RNAi agent. The objective here was to develop a non-toxic liposomal system of DTX and plasmid which can express PFKFB3 shRNA for inhibiting a key enzyme in the glucose metabolism for parental delivery. The co-delivery liposomal system was evaluated in lung cancer cells for its therapeutic effects. Further, A549 xenograft model was used to evaluate the therapeutic potential of the system and proof-of-concept was established through mechanistic studies to characterize the involvement of specific pathways in the therapeutic effectiveness of the combination. MATERIALS AND METHODS Materials Docetaxel (DTX) (LC Laboratories, Woburn, MA) was dissolved in dimethyl sulfoxide (DMSO) to prepare 1 mM stock solution and aliquots were stored at ?20C. Stock solutions were diluted to the desired final concentrations with medium just before use. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), N- (2,3-Dioleoyloxy-1-propyl) trimethylammonium bromide (DOTAB) and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethyleneglycol) (DSPE-PEG-2000) were obtained from Lipoid LLC, Germany. Dimethyldioctadecyl-ammonium bromide (DDAB) was purchased from SigmaCAldrich (St. Louis, Missouri). All the chemicals were used without any further purification. The mouse antibodies against – actin were purchased from Santa Cruz Biotechnology (Dallas, TX). The rabbit antibodies against Cdk2, Survivin, Bcl2, Cleaved Caspase 3, JNK, pJNK, p38 and p-p38 were purchased from Cell Signaling Technology (Danvers, MA). Secondary goat anti-rabbit or anti-mouse antibodies conjugated with horseradish peroxidase was bought from Santa Cruz Biotechnology (Dallas, TX). iScript? Advanced cDNA Synthesis Kit and SsoAdvanced? Universal SYBR? Green Supermixwere purchased from Bio rad (California, USA). Q-PCR primers for mouse PFKFB3 were purchased from Origene (Rockville, MD). PFKFB3 plasmid (pshPFKFB) was obtained from Origene Technologies (Rockville, MD). Cell Lines The cell lines, A549 (CCL- 185), H460 (HTB- 177) and HEK293 (CRL- 1573), were obtained from American Type Culture Collection (ATCC). All cell lines were maintained in Dulbeccos Modified Eagles medium (DMEM; Sigma Aldrich, St Louis, MO) nutrient mixture supplemented with 10% fetal bovine serum (FBS) from Invitrogen (Grand Island, NY) and antibiotic-antimycotic mixture.

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