Study Of The Reversal Effect And Mechanism On Multidrug Resistance Of Breast Cancer Cell By The Ginsenoside Rh₂

The mobility of breast cancer is increasing these years.Breast cancer is the second most common cause of cancer-related mortality in women. Breast cancer is one of the most common malignancies in women. Currently, the main treatments for breast cancer include surgery radiation therapy, chemotherapy and hormone therapy. Characteristics of breast cancer multidrug resistance has severely limited the implementation of chemotherapy.Cancer cell developing multidrug resistance (MDR) is one of the most serious clinical problems responsible for the failure ofcancer chemotherapy. Reststance cancers tend to manifest stimultanenous resistance to a number of structurally and functionally distinct anticancer agents, which is known as multidrug resistance (MDR), The main molecular mechanisms of MDR include. This phenomenon is known as MDR. Various mechanisms are involved in drug resistance in cancer, such as increased drug effhix or decreased drug influx,alteration in cell cycle checkpoints, failure of apoptotic mechanisms,changes in targeted enzymes,altered DNA repair and scavenging enzymes, etc. there are two strategies to overcome MDR:one is development of novel anticancer drugs which are sensitive to MDR cancer cells, the other is combination of MDR reversal agents with anticancer drugs to restore the senitivity of anticancer drugs on MDR cancer cells.The overexpression of P-gP(P-glycoprotein),which is encoded by MDR-1 (Multidrug resistance-1)gene, plays a key role in the resistant mechanisms. P-gP functions as an efflux pump to decrease the intracellular accumulation of a variety of lipophilic drugs, including paclitaxel.Extracellular matrix metalloproteinase inducer (EMMPRIN, also designated CD 147 or basigin), a surface glycoprotein of the immunoglobulin superfamily, has been demonstrated to stimulate production of MMPs in the tumor cells and in neighboring fibroblasts. EMMPRIN overexpression has been reported in many cancers and is shown to increase tumor cells invasion. MMPs (matrix metaloproteinases, MMPs), inuclding MMP-2 and MMP-9, play all important role in the degradation of basement membrane type IV collagen, which is associated with tumor cell invasion and metastasis. A role for MMPs in ovarian cancer development has been postulated based upon the observation that several members of the MMP family are up-regulated during ovarian cancer neoplastic progression. MMPs may play important role in the invasion and migration of tumor cells.The resistant cell line exhibited significantly elevated production of EMMPRIN and MMPs compared with its parental cells, and up-regulation of EMMPRIN and MMPs was induced by P-gp substrates in MDR cancer cells. In addition,P-gp substrates significantly enhanced the in vitro invasion abilities of MDR tumor cells. The facts above suggested that treatment of MDR tumor cells with P-gp substrates may produce a remarkable influence on malignant behavior that could adversely affect therapeutic outcomes. Whether MDR reversing agents which reverse MDR effectively have effects on tumor metastasis has few reported.Side effects of traditional Chinese medicine treatment of cancer is not only small and relatively wide pathway. In recent years a large number of studies have shown that many of the active ingredients of single herb and compound reversal in vitro in the role of MDR tumor cells. Ginsenoside (G) is the main active ingredient in Ginsen, Araliaceae. It has biological effects on anti-aging, anti-fatigue, anti-aggregation of platelets and radioprotection.Recently, more and more studies showed that G plays an important role on cancer prevention and treatment. G has many ways to anti-tumor by Directly retaining the growth of tumor cells, Inducing the cell apoptosis and differentiate, Inhibiting the metastasis of tumor through many channels, Enhancing the sensitivity of chemotherapy though reversing the resistance to chemical medicine and Adjusting the activity of many enzymes in cell or enhancing the immunity so that indirectly inhibit the growth of tumor cells. In this study, we want to investigate modulations of P-gP-mediated multidrug resistance of MCF-7/Adr by ginsenoside Rh2 in vitro.Ginsenoside for the role of MDR reversal, the study found ginsenoside Rg3 KBV20C can increase the sensitivity to multiple chemotherapeutic agents, and inhibited rhodamine 123 KBV20C cells from the outer row. Ginsenoside G-Rh2 in the experiment under the selected dose of B16-BL6 cells in spontaneous lung metastasis was significantly inhibited, and its mechanism and inhibition of matrix metalloproteinase-related, and show G-Rh2 ginsenoside toxic effects on normal cells low. However, ginsenoside G-Rh2 on reversing tumor drug resistance while the role of tumor invasion and metastasis research has not been reported, therefore, In this study, doxorubicin-resistant breast cancer cell lines (MCF7/AdrR) as target cells of ginsenoside G-Rh2 on the resistance reversal effect of resistant cells and resistant cells from apoptosis induced by G-Rh2 on the resistance invasion and metastasis-related gene expression, G-Rh2 ginsenoside clear reversal of the possible mechanisms of drug resistance.The results were as follows:1. We found that Gensenoside Rh2 at concentration as 5-40μmol/L had no cytotoxicity (the cell survival rate was over 90%), ADM at a concentration as 0.2-1μg/mL had no cytotoxicity (the cell survival rate was over 95%).2. Results showed that 5-40μmol/L of ginsenoside Rh2 and 1.0μg/mL of ADM combined, significantly decreased cell viability, cell survival, respectively ADM alone group was 63%,50%,31%,28%of cells by flow cytometry fluorescence intensity found,5-40μmol/L of ginsenoside Rh2 and 1.0μg/mL of ADM combined cell, the fluorescence intensity of intracellular ADM was significantly increased, intracellular ADM ADM fluorescence intensity are 1.09,1.55,1.61,2.37 times alone group, the differences were significant.3. Cells by fluorescence microscopy showed MCF7/AdrR the role of GS-Rh2 was more obvious after the morphological changes of apoptosis, ADM+Rh2 different concentrations significantly reduced the number of nuclei, a large shrinkage deformation of the nucleus, chromatin condensation, stain showed dense fluorescent particles, formation of apoptotic bodies,especially large dose of ADM and Rh2 apoptosis of the most obvious combination group.4. GS-Rh2 exhibited growth inhibition efect and induced apoptosis on MCF7/AdrR cell line in vitro.The combination of ADM and Gensenoside Rh2 lead to apoptosis of MCF-7/Adr cels(47.08%)detected by flow cytometry, which was much higher than control(8.51%)and ADM (15.20%), Rh2+ADM effect of each concentration group appeared in MCF7/AdrR cell lines were induced G0/G1 arrest, block cells from G1 to S phase of the transformation, with the increasing concentration of Rh2 role content of G0/G1 phase cells increased, S decrease of cells, G2/M phase compared with the control group no significant changes.5. Rh2 group and the ADM group, Fas, Bax, Bcl-2, Bcl-xL protein expression compared with the control group showed no significance juice system. ADM+Rh2 role in MCF7/AdrR different cell concentrations to Fas, Bax protein increased, Rh2 concentration of 40μmol/L increased expression of the most obvious time, Bcl-2 protein expression decreased, Bcl-xL protein expression in drug showed no obvious change.6. First, RT-PCR and Western blot results showed that 1.0μg /mLADM role in MCF7/Adr cells, compared with the control group, cell P-gp, EMMPRIN and MMP1, MMP2, MMP9protein and mRNA levels, and then we and observed ginsenoside Rh2on the cell P-gp, EMMPRIN, and MMP1, MMP2, MMP9 expression. The results showed that when ginsenoside Rh2 and ADM combined, ginsenoside Rh2 also inhibited the ADM on the cell the protein and mRNA levels increased and ginsenoside Rh2 on the protein expression decreased with ginsenoside Rh2 concentration dependent in a concentration dependent sex.7. The role of ginsenoside Rh2 on MCF7/Adr cells, the transwell chamber assay and found that different concentrations of ADM+Rh2 group and control group cells across Matrigel covered or not covered by Matrigel membrane holes are the reduction of the number; and compared with the control group ginsenoside Rh2 inhibited the cell invasion and metastasis MCF7/Adr, the inhibition rates were 16%,26%,49%,74% and 18%,23%,52%,78%. The ADM group compared with control group, MCF7/Adr cells across Matrigel covered or not covered by an increase in the number of Matrigel membrane hole.These results suggest that:1. Ginsenoside Rh2 can reverse drug resistance to ADM in MCF-7/Adr cells significantly in vitro.The resistance reversal effect with P-gp related to ginsenoside Rh2 may directly or indirectly acting on the P-gp,its dysfunction or inactivation,Rh2 as calcium channel blockers compete with anticancer drugs combination of P-gp site,thus inhibiting the role of the transmembrane pumps that efflux of anticancer drugs decreased the cell, increased intracellular concentration of drug resistance reversal.2. GS-Rh2 exhibited growth inhibition efect and induced apoptosis on MCF7/AdrR cell linein vitro.3. Apoptosis of MCF7/AdrR cells induced by GS-Rh2 maybe related with arresting cell cycle,up-regulating Fas,Bax protein expressionand down-regulatingmutated Bcl-2 protein expression.4. Gensenoside Rh2 (GS-Rh2) not only reversed MDR effectively,but also inhibited the up-regulation of P-glycoprotein, EMMPRIN, MMP2 and MMP9 by ADM, reduced cells in vitro invasive and migrative abilities. Gensenoside Rh2 could inhibit tumor invasive and migrative ability by down-regulation the expression of MMP-2 and MMP-9 in MCF7/Adr cancer cells.