| BackgroudBreast cancer is one of the most common malignants tumor in women of the world. The incidence rate increased year by year in our country. A long-term follow-up research about the correlation of cancer and depression demonstrated that the co-morbidity proportion of patients with breast cancer and depression is up to10%-30%due to the special part of the body (breast). Depressive disorder had a strong influence on the compliance and prognosis of patients, which resulted in the decline of life quality. Furthermore, depressive disorder might affect drug efficacy by interfering pharmacokinetic precess. Therefore anti-depressants such as fluoxetine have been used in cancer patients with depression. Clinical study suggested that cancer chemotherapy patients treated by fluoxetine lead to the improvement of depression and the enhancement of life quality.Multidrug resistance (MDR) is the main reason for chemotherapy failure in clinical chemotherapy. MDR is a phenomenon that cancer cells develop a cross-resistant phenotype against several unrelated drugs that differ widely with respect to molecular structure and target specificity. MDR includes intrinsic and acquired drug resistance. The mechanism of MDR involves four aspects. First and the most important, activation of transporter proteins (P-glycoprotein, multidrug resistance-associated protein (MRP)); second, activation of the enzymes of the glutathione system especially glutathione S transferase π isozyme (GST-π), sequestration of the drug in intracellular vesicles; third, mutations of p53gene, activation of bcl-2gene; fourth, mutations of the genes coding for topoisomerases Ⅱ, enhancement of DNA repair.MDR is a major barrier for breast cancer chemotherapy. The enhancement of chemotherapy sensitiveness is a key to overcome MDR. MDR reversal agents come to the third generation at present, from first generation such as calcium ion channels blocker verapamil, PKC inhibitor, tamoxifen to second generation (dexverapamil, dexniguldipine, PSC833) and to third generation such as tetrandrine. Although so many reversal agents are certificated to reverse MDR, clinical application is limited due to serious adverse drug reactions and toxicity. Research and development of new MDR reversal agents is a hotspot in cancer pharmacology.Fluoxetine (FLX), the selective serotonin reuptake inhibitors (SSRI) was used to treat depression effectively in clinical. It improved cancer patients’immunity, life quality and extend their live time. Further more FLX was reported to be a new highly effective chemosensitizer.At present, TAC, AC combination with FLX has become the routine chemotherapy in breast cancer treatment. However, no systemic pharmaocological investigation has been conduceted up to now. The project would study the pharmacological effect and mechanism underlying common anticancer agents adriamycin (ADM) and paclitaxel (PTX) with FLX interaction and hope to supply any pharmacoligcal evidence for clinical application.This paper aims to study the effect of FLX reverse MDR in resistant breast cancer cells from two aspects:one is to analysis the pharmacodynamics of anticancer drugs ADM and PTX with or without FLX in breast cancer cells MCF-7/ADM and MCF-7; the other is to research the proteins expression levels of P-gp, GST-π, p53, Bcl-2and mRNA expression level of MDR1, and to interpret the possible mechanism of FLX acts as a chemosensitizer.Objectives1By detection the proliferation inhibition and apoptosis effect of ADM/PTX alone or with FLX in MCF-7/ADM and MCF-7cells, we investigated the pharmacology function of FLX on cancer cells.2By assay of P-gp, GST-π, p53, Bcl-2proteins expression, MDR1mRNA expression, we investigated the possible mechanism involved in drug transport and apoptosis of FLX modulates MDR and enhancement chemotherapy sensitivity in breast cancer cells.Methods1MTT assayed of cells proliferation inhibitionIn MTT test, cells were seeded into96-well plates and treated with various doses of FLX (2.5-80μg/ml in MCF-7/ADM and MCF-7cell lines), ADM (2.5-80μg/ml in MCF-7/ADM cells;0.125-4μg/ml in MCF-7cells), PTX (0.5-16μg/ml in the two cell lines) alone or FLX-ADM, FLX-PTX (5μg/ml FLX with various concentrations of ADM or PTX the same as it treated alone) combination for72h. Results were plotted as percent of survival and concentration-response curves were fitted in order to determine the50%effective concentration (IC50). According to the IC50values of ADM or PTX alone and with FLX, we calculated the reversal fold (RF) of FLX. The statistical significances were measured by t-test.2Flow cytometry detected apoptosisAccording to the results of MTT analysis, we chose1,4μg/ml respectively as the low (PTX(1)), high (PTX (h)) combination concentration of PTX in MCF-7/ADM and MCF-7cells;5,20μg/ml respectively as the low (ADM(1)), high (ADM (h)) concentration of ADM in MCF-7/ADM cells,0.5,2μg/ml respectively as the low (ADM(1)), high (ADM (h)) concentration of ADM in MCF-7cells. In order to assess the effect of combination of FLX, cells were exposed to an equivalence concentration of ADM or PTX in addition with5μg/ml of FLX. The two cell lines were grouped by10treatments:control、PTX(1)、PTX(h)、FLX-PTX(1)、FLX-PTX(h)、ADM(1)、 ADM(h)、FLX-ADM(1)、FLX-ADM(h)、FLX.24h later, cells were stained with Annexin V-FITC and PI, then analyzed on an EPICS XL flow cytometry. The statistical significances were measured by one-way ANOVA and t-test.3Western blot assayed of proteins expression MCF-7/ADM or MCF-7cells were treated the same as above for72h. The total protein was extracted and P-gp, GST-π, p53, Bcl-2were assayed by Western blot. The statistical significances were measured by one-way ANOVA and t-test.4RT-PCR, qRT-PCR detected the MDRl gene expressionMCF-7/ADM or MCF-7cells were treated the same as above for48h. The total RNA was extracted and the mRNA of MDR1were assayed by RT-PCR and qRT-PCR. The statistical significances were measured by one-way ANOVA and t-test.Results1Proliferation inhibited by drugsIC50values of FLX were25.34±1.3μg/ml in MCF-7/ADM cells and17.56±0.98μg/ml in MCF-7cells. Inhibition rates of FLX in MCF-7and MCF-7/ADM were less than10%(5.43±2.33%and7.53±0.98%, respectively) at the concentration of5μg/ml, so we chose5μg/ml as the combination concentration. FLX enhancement the inhibition rate of ADM and PTX in the two cell lines, especially at the low concentrations. Concretely, with ADM less than20μg/ml in MCF-7/ADM cells and2μg/ml in MCF-7cells, PTX under4μg/ml in the two cell lines, combination with FLX showed a significantly increase in the proliferation inhibition rates.ADM combination treatment with FLX generated a statistically significant5.03-fold reduction in IC50in MCF-7/ADM cells, from13.62±1.44μg/ml alone to2.71±0.42μg/ml with FLX (P<0.001); but had slight effect on MCF-7cells with the IC50values of0.53±0.12μg/ml alone and0.45±0.04μg/ml with FLX (P=0.338). The data indicated that MCF-7/ADM cells were25.7-fold more resistant to ADM than MCF-7cells (P<0.001).FLX-PTX combination had statistical differences of reversal effect on the two cell lines. IC50values were3.30±0.12μg/ml alone and2.59±0.11μg/ml with FLX in MCF-7/ADM cells (P=0.002),2.11±0.37μg/ml alone and1.26±0.16μg/ml with FLX in MCF-7cells (P=0.019). IC50value of PTX alone in MCF-7/ADM cells was higher than in MCF-7cells (P=0.006).2Apoptosis enhancement induced by FLX combinationThe results demonstrated that FLX alone had little effect on cell apoptosis (3.17 ±0.70%and3.84v0.31%respectively), but significantly increased apoptosis rate while FLX-ADM or FLX-PTX combination. In MCF-7/ADM cells, apoptotic proportion increased from5.63±0.07%to19.15±3.35%treated by5μg/ml ADM with5μg/ml FLX (P=0.020); from35.53±1.65%to49.00±6.05%treated by FLX-ADM(h)(P=0.025); increased5.73%and21.90%respectively by treatment with FXL-PTX(1) and FLX-PTX(h), showed a significant enhancement in apoptosis rate (P=0.001, P<0.001respectively). Similar increase was also observed in MCF-7cells.3Levels of p53, Bcl-2, P-gp, GST-π proteins expression modulated by FLX3.1p53expressed in MCF-7/ADM and MCF-7cellsIn MCF-7/ADM cells, with the exception of FLX alone group (P=0.953), other drug treatment groups increased p53level remarkly compared with the control group (P<0.001respectively). The combination groups up-regulated the expression of p53in a concentration-dependent manner, significantly induced p53expression compared with their alone groups.FLX (5μg/ml) alone increased p53levels in MCF-7cells. The similar results were got in MCF-7cells, much better induced effect were got by treatment with PTX alone or FLX-PTX combination groups than ADM treatment groups.3.2Bcl-2expressed in MCF-7/ADM and MCF-7cellsIn MCF-7/ADM cells, FLX alone had no effect on the expression of Bcl-2protein compared with control group. Bcl-2expression level increased by treatment with ADM/PTX alone but decreased by combination with FLX. Especially in PTX groups, Bcl-2level rose to more than2-fold treated by PTX alone but sharp down to less than50%compared with the control group by FLX-PTX combination.In MCF-7cells, the drugs treated groups down-regulated Bcl-2level with the exception of the FLX alone and ADM(1) alone groups. FLX-ADM or FLX-PTX combination groups significantly down-regulated Bcl-2expression level (P<0.001respectively), but not so pronounced as it in MCF-7/ADM cells.3.3P-gp expressed in MCF-7/ADM and MCF-7cellsIn MCF-7/ADM cells, FLX alone group and ADM with the presence or absence of FLX groups had no effect on the expression of P-gp protein compared with the control group. PTX alone up-regulated P-gp level to3.6fold of the control group, While FLX-PTX combination down-regulated P-gp level significantly to less than30%of control group, showed a statistical differences.In MCF-7cells, Neither ADM alone groups or combination groups changed the P-gp level in comparation with control group. There was no significant difference between ADM(1) alone and FLX-ADM(1) group (P=0.474), but decreased in FLX-ADM(h) group compared with ADM(h) group (P=0.015). Compared with the control group, PTX(1) group had no effect on P-gp level while PTX(h) induced P-gp expression. FLX reduced P-gp expression to68.22%of the control group in FLX-PTX(1) group and reversed the overexpression of P-gp in FLX-PTX(h) group,(P<0.001, P<0.001respectively VS their alone groups).3.4GST-π expressed in MCF-7/ADM and MCF-7cellsIn MCF-7/ADM cells, GST-π protein had no change in the FLX alone group compared with the control group (P=0.991), GST-π level was decreased in a concentration-dependent manner by treatment with PTX or ADM alone (all P<0.001), the similar with Bcl-2expression in MCF-7/ADM cells by FLX combination. GST-π was undetected in MCF-7cells.4Expression of MDR1geneMDR1mRNA was assayed using RT-PCR analysis. After48hours of treatment with ADM or PTX alone, the level of MDR1gene was enhanced strikingly as the increasing of drug concentration in MCF-7/ADM cells. FLX alone did not change compared with control, but FLX-PTX combination down-regulated its expression at both concentrations. FLX-ADM combination came to an opposite results. MDR1gene was undetected in MCF-7To validate the results of RT-PCR, qRT-PCR was also performed. The results were accordant. Comparison to ADM treated alone, the expression of MDR1was increased2-fold and5-fold respectively by FLX combination with low or high concentrations of ADM. FLX-PTX combination reduced to68%(low) and50%(high) in the expression of MDR1gene. MDR1gene expressed200-fold in MCF-7/ADM cells than in MCF-7cells, which made it negligible for the MDR1mRNA expression in MCF-7cells.Conclusion1FLX synergized with ADM/PTX by enhancement of the proliferation inhibition and inducing apoptosis effects in MCF-7/ADM and MCF-7cells with none cytotoxicity, as a potential chemosensitizer.2The reversal mechanisms of FLX involve in affected indirectly or indirectly the drugs efflux pump P-gp, down-regulation the expression of Bcl-2and GST-π, up-regulation p53level. |
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