| Multidrug resistance (MDR) is one of the main reasons for failure of cancer therapy. The MDR mechanism is complex and not fully understood. The most common factors include that over-expression of ATP dependent efflux pumps, reduction of medicine absorption, and/or alterations in survival or apoptotic pathways. Over-expression of P-gp is the most common reason of MDR. A range of agents, such as verapamil and cyclosporin, that can reverse the MDR phenotype and restore drug sensitivity to cancer cells has been developed. However, most of these agents have been proven to be intrinsic toxic or accompanying with side-effects during treatment. New reliable reversal agents with less toxicity and better effects have been always the aim for the researchers.Hyaluronaic acid (HA) is a large glycosaminoglycan with a molecular weight ranging from105to107Da. HA not only is an important structural component of extracellular matrices but also interacts instructively with cells in embryonic development, healing processes and inflammation. HA oligosaccharides (o-HAs) can be generated by enzymatic degradation of HA. The molecular weoght is normally less than104Da. O-HAs perform even opposite biological activities compared to HA. O-HAs can modulate growth and cell survival process of cells. They can also sensitize MDR cancer cells to cytotoxic drugs. In this project, o-HAs were used to study the mechanism of MDR reversal effect. The effect of o-HAs on the modulation of P-gp function and expression were studied. 1Preparation of single polymerized o-HAs.Conditions of HA hydrolysis catalyzed by hyaluronidase (HAase) were optimized as follows:10g/L of HA,1.5×106U/L of HAase, pH5.0, temperature50℃. Bio-gel P6and G-10chromatography were used to purify the digenstion mixture. Samples were characterized by mass spectrometry and confirmed to be HA tetrasaccharide (o-HA4), HA hexasaccharide (o-HA6) and HA octasaccharide (o-HA8).2Effects of o-Has on tumor MDRK562/A02and MCF-7/Adr cells treated with adriamycin (ADR) were used as the control group. The MDR fold of K562/A02and MCF-7/Adr cells was98.90and166.03. The cell growth inhibition of K562/A02cells and MCF-7/Adr cells was assayed using MTT method. When the concentration of o-HAs at100μg/mL, the RF of K562/A02cell were3.2429、31251and4.2807, respectively, while those of MCF-7/Adr cells were2.8、2.95and3.08. The intracellular accumulation of ADR the intracellular accumulation and efflux levels of Rh123were measured. When the concentration of o-HAs at100μg/mL, the intracellular accumulation of ADR in K562/A02cell were increased2.8-.2.95and3.08, respectively. The intracellular accumulation of ADR in MCF-7/Adr cells were increased2.13、2.23and2.32, respectively. The intracellular accumulation of Rh123in K562/A02cell were increased2.34、2.45and2.46, respectively, while those in MCF-7/Adr cells were3.31、3.47and3.0. The efflux levels of Rh123were reduced at the same time in both cell lines.3Mechanism of MDR reversal effect of o-HAsTo examine the effects of o-HAs on intracellular ATP levels,K562/A02cells and MCF-7/Adr cells were treated with various concentrations of o-HAs solutions. ATP was determined using a luciferase assay. When the concentration of o-HAs at100μg/mL, ATP was decrased in K562/A02cells by29.76%、30.06%and32.6%, respectively, and48.08%、51.88%and51.07%in MCF-7/Adr cells. P-gp-ATPase activity was studied by determining different release levels of ingoranic phosphate, o-HAs activated the P-gp-ATPase activity in the ratio of4.32、4.22and3.81in K562/A02cells, and2.93、2.86and2.87in MCF-7/Adr cells. The activity of PKC was determined according to Assay for Non-Radioactive Detection of PKC. o-HAs decreased the activity of PKC by54.36%,54.17%and62.11%in K562/A02cells, and58.86%,57.71%and59.12%in MCF-7/Adr cells. The expression levels of PTEN and AKT was studied using Western blot. Cells were esposed to o-HAs at the concentraton of100μg/mL. The expression levels of PTEN was increased with4.31、6.31and5.01in K562/A02cells, and2.31,2.54and2.58in MCF-7/Adr cells. The expression levels of Akt were decreased by45.38%,51.84%and61.32%in K562/A02cells, and43.28%,56.04%and54.35%in MCF-7/Adr cells. The level of MDR1mRNA was evaluated by RT-PCR. The levels of MDR1mRNA were decreased by13.49%,24.6%and27.78%in K562/A02cells, and22.23%,29.36%and33.78%in MCF-7/Adr cells.Results from this project:1Differences of o-HAs treatments on MCF-7/Adr and K562/A02cell lines were obtained. An increase of Rh123inside MCF-7/Adr and K562/A02cells and decrease of efflux effect were observed when cells were treated with o-HAs.2A reduced activity of PKC and higher level of P-gp in K562/A02and MCF-7/Adr cells were observed. The P-gp-ATPase activity in membrane of K562/A02and MCF-7/Adr cells was stimulated after treated with o-HAs. The activity of PKC significantly decreased after the o-HAs treatment.3O-HAs can promote the expression of PTEN while inhibiting that of Akt. O-HAs can inhibit the expression of MDR1mRNA. These data were in good agreement with the observation that the activity and expression of P-gp were inhibited.4MDR reversal effects of o-HA8and o-HA6are better than that of o-HA4.Single polymerized o-HAs were prepared to study the possible mechanism of MDR reversal effect. O-HAs can reverse tumor MDR through modulating the activity and expression of P-gp. The study has laid the foundation for the development of o-HAs as potential MDR reversal drugs. |
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