| Background and ObjectiveChemotherapy is one of the classical treatment for malignant tumor in clinic. However, as the conduct of the treatment to the tumor or its different reactivity to chemotherapeutic drugs, many tumors develop resistance to chemotherapeutic drugs, sensitivity reduction, chemotherapy-induced bone marrow suppression, neurotoxicity, cardiovascular toxicity and other side effects, which not only seriously affects people's the quality of life, but also limits the chemotherapy drug application and increases the difficulty of clinical cancer treatment. Therefore, it is an urgent issue to overcome chemotherapy resistance , reduce drug side effects and improve the effect of chemotherapy.Recent studies about how to overcome chemotherapy resistance and improve the effect of chemotherapy have suggested that UPS inhibitor is a new anti-cancer drug, which can promote tumor cell into apoptosis, enhance drugs chemo-sensitivity and overcome its chemoresistance when it was used independently or combined with other anti-cancer drugs. And it was reported that proteasome inhibitor MG132, as an anti-cancer drugs have its own potential for separated or combined use with other chemical reagent. Ups inhibitors have been used in clinic.Multidrug resistance of tumors is related to energy metabolism of tumor cells, since energy supply from ATP is required when the multi-drug resistance protein transport chemotherapeutic drugs. Studies reported that regulating the energy supply to tumor cells can increase to chemosensitivity. Whether it can elevated the efficacy when proteasome inhibitor was used with chemotherapy drugs in different conditions of energy supply ,especially in the condition of low energy supply , which was reported seldom.In this study, D-glucose (D-glucose, D-G) or L-glucose (L-glucose, L-G) were added into glucose-free medium to change the energy supply of tumor cells ,then observe the growth inhibition effect of MCF-7 and K562 cells when they were treated with UPS inhibitor MG32 and broad-spectrum anti-cancer drugs Paclitaxel in two different conditions of energy supply, and then explore its possible mechanism.Materials and methods1. Materials:1.1 Cell lines: K562 cell line, purchased from the Institute of Hematology, Chinese Academy of Sciences。MCF-7 cell line, obtained from Experimental Center of Guangzhou Medical College.1.2 Reagents : Paclitaxel ( Hainan Sinochem Co., Ltd. a joint production of the pharmaceutical industry, the national medicine accurate H20057065); D-G(Guanghua Chemical Factory in Shantou City); L-G (Shanghai Electrical and Mechanical Co., Ltd. Western Region); MG132 (Alexis);Paclitaxel was dissolved in NS and preserved at- 20℃, MG132 was dissolved in DMSO and preserved at- 20℃.2.Methods:2.1 Cell culture : K562 cells and MCF-7 cells were maintained in RPMI1640 complete medium containing 2 g /L glucose supplemented with 10% fetal calf serum (FCS).K562 cells and MCF-7 cells were passaged one time every other day and every two days with 0.25% trypsin (containing EDTA) ,reapectively. and cultured in a humidified atmosphere 37℃5% CO2 incubator. Glucose-free complete medium supplemented with 10% fetal calf serum were added D-G or L-G as D-G and L-G complete medium , and were made the final concentration as 2g / l, respectively. logarithmic growth phase cells were used for experimental as follows: cells were passaged and centrifugated ,then we adjusted the cell concentration and re-suspended cells in the D-G or L-G complete medium mentioned above ,and the do experiment according to the experimental groups .2.2 Experimental groups: Groups were divided into negative control group, Paclitaxel group, MG132 group and Combined group(Paclitaxel+MG132). The concentration of MG132 and Paclitaxel is different depending on the experiment. The concentrations of Paclitaxel and MG132 range from 20nM / l to 10μM /l and 0.625μM /l to 10μM /l, respectively.2.3 Cells viability detection :MTS assay was used to detect the effect of cells viability of K562 and MCF-7, which were treated with MG132 1μM and Paclitaxel in the D-G and L-G culture conditions. Then a suitable concentration was determined for follow-up flow cytometry analysis of cell cycle and apoptosis and so on.2.4Study of the effect of cell death on K562 which were treated with MG132 and Paclitaxel : The cell death of each group in two different conditions of energy supply was detected with PI staining and FCM ,respectively.2.5 Study of the effect of the expression of Bax and PARP on K562 cells which were treated with MG132 and Paclitaxel : the expression level of Bax and PARP of each group in two different conditions of energy supply was detected by Western blotting.2.6 Study of the effect of cell cycle of K562 which were treated with MG132 and Paclitaxel :A suitable concentration was chosen according to the above results, and the cell cycle of K562 cells in two conditions of energy supply was detected by flow cytometry (FCM).Results1. MTS assay was used to detect the cell viability ,and then a suitable cell lines was sifted. 1.1Cells relative viability were 67.96%～52.24% after MCF-7 cell were treated with 2.5μM～10μM MG132 for 24 hrs, When compared with control group, the difference was significantly (p<0.05).No significant inhibitory effect was observed when the concentration of MG132 was lower than 2.5μM.1.2 Cells relative viability was decreased when Paclitaxel concentration increase , The dose-response relationship was obvious. Cells relative viability of MCF-7 in the D-G and L-G conditions was 49.05%,49.09%,and 47.57%,35.09% after cells were treated with, 5μM, 10μM Paclitaxel, respectively. When compared with control group, the difference was significant (p<0.05); Cells relative viability of K562 in the D-G condition was 77.10%,63.35%,54.61%, after cells were treated with, 1μM,5μM,10μM Paclitaxel, respectively. When compared with control group, the difference was significant (p<0.05); Cells relative viability in the L-G condition was 59.06%,50% after cells were treated with 5μM, 10μM Paclitaxel, respectively . as compared with the control group was statistically significant (p <0.05).1.3The combination group compared with the single treatment group were not statistically significant when MCF-7 cells were treated with MG132 and Paclitaxel concurrently in both D-G and L-G conditions . whereas cells relative viability of K562 cells in the D-G condition was 50.97%, 29.63%, 15.41% after cells were treated MG132 1μM with 1μM, 5μM, 10μM Paclitaxel, respectively. as compared with the control group was statistically significant (p <0.05).2. 2 The effect of cell death on K562 which were treated with MG132 and Paclitaxel:2.1 Cells which were treated with MG132 and Paclitaxel were detected by FCM :2.1.1The apoptosis rate of the combined group in L-G conditions was significantly elevated when K562 cells were treated with MG132 1μM and Paclitaxel 5μM, as compared with the single drug group was statistically significant (p <0.05),and it is much higher than that in the D-G conditions(p <0.05);The apoptosis rate of Paclitaxel in the L-G conditions is pronounced, as compared with the control group was statistically significant (p <0.05). The apoptosis rate of the combined group in D-G conditions also increased,as compared with the single drug group was statistically significant (p <0.05). 2.1.2The apoptosis rate also increased significantly in both conditions when K562 cells were treated with MG132 1μM and Paclitaxel 20nM,1μM,and the time-dependent effect is obvious; The effect of apoptosis is markedly elevated in the L-G conditions.2.2 The cellular morphology were observed after PI staining:The cell death increased significantly when K562 cell were treated with MG132 0.5μM and Paclitaxel 20nM in the L-G condition, moreover, the necrotic cell increased markedly; and that the cell death elevated markedly when K562 cell were treated with MG132 1μM and Paclitaxel 200nM in the L-G condition .The cell death of MG132 treatment group and the combined treatment group increased obviously.3. The effect of Bax and PARP on K562 cells which were treated with MG132 and Paclitaxel :The accumulation of Bax was significantly increased when the cells were treated with MG132 alone in the D-G condition, and there is also accumulation of Bax in the L-G condition.The fragment of PARP was elevated in the combined group in D-G condition , no significant changes can be observed in the combined group in L-G condition.4 The effect of cell cycle of K562 which were treated with MG132 and Paclitaxel:The cell cycle mainly arrested in the G2 / M Phase when K562 cells were treated with Paclitaxel 20nM and MG132 0.5μM or 1μM in the L-G condition, respectively .. Moreover, With the time extended from 24h to 48h . the percent of G2-M Phase in the combined group is elevated ,and the peak of apoptosis in the combined group also increase.Conclusion1.After reducing energy supply , the cell cycle mainly arrested in the G2 / M Phase when K562 cells were treated with low concentrations of Paclitaxel (20nM) and low doses of MG132(0.5μM or 1μM) , moreover , the apoptosis and death of K562 increase obviously. 2. Decreasing energy supply can enhance the sensitivity of K562 cell to MG132 and Paclitaxel . |
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