Expression Of Multidrug Resistance Protein Research Articles

Using Caffeine and Free Amino Acids To Enhance the Transepithelial Transport of Catechins in Caco-2 Cells.

Catechins are well-known to possess health-promoting functions. The interaction of the catechins with other components in tea could alter their absorption and efflux. This study investigated whether the absorption of catechins is affected by caffeine and amino acids using the Caco-2 monolayer cell model. We found that (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC) were all actively effluxed. Co-transportation of EGCG, ECG, or EC with caffeine, theanine, serine, or glycine increased their apparent permeability coefficient [ Papp(AP-BL)] value by 3.42-5.40- fold, 1.19-5.75-fold, and 1.55-8.01-fold, respectively. Meanwhile, their efflux ratio values were significantly decreased. Moreover, the expression of multi-drug resistance protein 2 (MRP2) after 3 h of incubation with either 50 μM EGCG or 50 μM EC was elevated by 1.58- and 2.98-fold, respectively, while 50 μM ECG had no significantly effects. In addition, the expression of P-glycoprotein (P-gp) after treatment with either 50 μM EGCG, 50 μM ECG, or 50 μM EC was enhanced by 1.53-, 1.63-, and 1.80-fold, respectively. The addition of either caffeine or any one of the three amino acids decreased the expression of both MRP2 and P-gp induced by EGCG, and the expression of P-gp induced by ECG or EC also decreased. In contrast, only glycine decreased the expression of MRP2 induced by EC. Taken together, our data indicated that caffeine and theanine, glycine, or serine in tea might increase the absorption of catechins by the selectively suppressed expression of the efflux transporters induced by catechins.

LINC00511 knockdown prevents cervical cancer cell proliferation and reduces resistance to paclitaxel

Cervical cancer (CC) is one of the most common female malignancies in the world. Although paclitaxel (PTX) is a critical chemotherapy agent for the treatment of CC, its treatment outcome is limited by the development of drug resistance. The present study aims to define the role of long non-coding RNA (lncRNA) LINC00511 in the progression of CC with the involvement of cell proliferation, apoptosis and resistance to PTX in Hela/PTX cells. CC and adjacent normal tissue samples were collected from 84 patients with CC, and used to determine LINC0051 expression. PTX-resistant Hela/PTX cell line was constructed, in which LINC0051 was overexpressed or silenced to further investigate the effect of LINC00511 on PTX-resistant Hela/PTX cell viability, proliferation, migration, invasion, cell cycle, apoptosis and resistance of CC cells to PTX. The expression of Bcl-2, Bax, cleaved-caspase-3, matrix metalloproteinase (MMP)-2, MMP-9, multidrug resistance protein 1 (MRP1) and P-glycoprotein (P-GP) was also assessed. High-expression of LINC00511 was found in CC with its close association with the tumor stage, tumor size and lymph node metastasis. After silencing LINC00511 expression, the expression of MRP1, P-GP, Bcl-2, MMP-2 and MMP-9 was decreased, while Bax and cleaved-caspase-3 increased with more CC cells arrested at G1 phase. Furthermore, silencing of LINC00511 could suppress cell resistance to PTX, cell viability, cell proliferation, migration and invasion yet promoted cell apoptosis in PTX-resistant Hela/PTX cells. Collectively, our findings demonstrate that silencing of LINC00511 could inhibit CC cell resistance to PTX, cell proliferation, migration and invasion, and promote cell apoptosis in CC. Silencing of LINC00511 provides a novel therapeutic target for CC.

Human recombinant FSH induces chemoresistance in human breast cancer cells via HIF-1α activation†.

Breast cancer patients under 40 years of age who are candidate to chemotherapy with alkylating drugs may undergo controlled ovarian stimulation (COS) with recombinant human follicle-stimulating hormone (rhFSH) in order to get fertility preservation by mature oocyte cryostorage. The direct effect(s) of exogenous rhFSH on the chemosensitivity of breast cancer is currently unknown. To clarify this issue, we incubated four different breast cancer cell lines with rhFSH (10 IU/L, 24 h) and then we exposed them to doxorubicin (DOX) or cyclophosphamide (CPA). The effect(s) of rhFSH on human breast cancer cells treated with DOX or CPA was measured in terms of (1) cell viability, (2) cytotoxicity, (3) multidrug resistance (MDR) genes and proteins expression and activities, and (4) hypoxia-inducible factor 1-alpha (HIF-1α) activation. Pretreatment with rhFSH significantly increased the viability of breast cancer cells after treatment with DOX or CPA, and reduced the lactate dehydrogenase leakage and reactive oxygen species production. Moreover, after preincubation with rhFSH, the MDR proteins (Pgp, MPR1, and BCRP) expression and activity resulted upregulated and the HIF-1α pathway activated. In addition, the use of a widely used HIF-1α inhibitor, the 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), prevented the rhFSH effect on the onset of MDR. Taken together, these observations suggest that a short exposure to rhFSH induces chemoresistance to DOX and CPA in human breast cancer cells via HIF-1α activation.

Brain region-specific regulation of histone acetylation and efflux transporters in mice.

Multidrug resistance protein 1 (MDR1) and breast cancerresistance protein (BCRP) protect the brain by restricting the passage of chemicals across the blood-brain barrier. Prior studies have demonstrated the epigenetic regulation of MDR1 and BCRP in cancer cells treated with histone deacetylase (HDAC) inhibitors that enhance histone acetylation and gene transcription. In the present study, we tested the in vivo effects of two HDAC inhibitors, valproic acid (VPA; 400 mg/kg) and apicidin (5 mg/kg), on Mdr1 and Bcrp transporter expression in brain regions of adult male mice injected intraperitoneally daily for 7 days. VPA increased Mdr1 protein expression in the striatum (70%) and Bcrp protein in the midbrain (30%). Apicidin enhanced striatal Mdr1 protein (30%) and hippocampal Bcrp protein (20%). Transporter induction correlated with increased histone H3 acetylation in discrete brain regions. In conclusion, HDAC inhibitors upregulate transporter proteins in vivo, which may be important in regulating regional xenobiotic disposition within the brain.

Over-expression of FSIP1 promotes breast cancer progression and confers resistance to docetaxel via MRP1 stabilization

Fibrous sheath-interacting protein 1 (FSIP1) functions centrally in breast carcinogenesis and progression, although its exact role remains to be clarified. Therefore, we sought to establish a correlation between the clinico-pathological features of breast cancer and FSIP1 expression in breast cancer tissues, as well as to validate its role in tumor progression and chemo-resistance. We analyzed FSIP1 expression in the breast cancer and para-tumor tissues by immunohistochemistry. We performed MTT, Caspase-Glo 3/7 Assay, Annexin V staining, wound healing and trans-well assays to evaluate cellular apoptosis, proliferation, migration and invasion in FSIP1 knockout and wild-type breast cancer cell lines. Additionally, we examined the effects of FSIP1 on docetaxel sensitivity in a nude mice model transplanted with control or FSIP1 knockout breast cancer cells, and also evaluate its role in tumor metastasis. FSIP1 and MRP1 interaction was determined by co-immunoprecipitation and mass spectrometry. We found that breast cancer cells and tissues consistently demonstrated elevated FSIP1 expressions, which correlated with poor overall survival. Notably, patients with high FSIP1 expression in their tumors undergoing docetaxel neoadjuvant chemotherapy had shorter disease-free survival. FSIP1 knockout in breast cancer cells significantly increased their sensitivity to docetaxel both in vitro and in vivo. Mechanistically, FSIP1 bound to the multidrug resistance protein 1 (MRP1) and stabilized it, and knocking out FSIP1 decreased MRP1 expression and increased cellular docetaxel accumulation. In sum, FSIP1 promotes breast carcinogenesis and mediates docetaxel resistance, and may serve as a novel target in the development of breast cancer therapies.

AS602801 Sensitizes Ovarian Cancer Stem Cells to Paclitaxel by Down-regulating MDR1.

AS602801, an anti-cancer stem cell (CSC) candidate drug, sensitizes ovarian CSCs to paclitaxel and carboplatin by reducing the expression of survivin, an anti-apoptotic protein. The aim of the study was to examine the effect of AS602801 on the expression of multi drug resistance protein 1 (MDR1). Using two ovarian CSC lines, A2780 CSLC and TOV-21G CSLC, mechanisms other than survivin down-regulation were examined by comparing the effects of AS602801 and YM155, an inhibitor of survivin. After screening for the expression of ATP-binding cassette (ABC) transporters with or without AS602801 treatment, the sensitivity of cells to paclitaxel, carboplatin, and cisplatin was examined following knockdown of the ABC transporter. The combinational effect of AS602801 on paclitaxel was less dependent on survivin than the effect on carboplatin. AS602801 reduced the expression of MDR1, an ABC transporter. Knockdown of MDR1 sensitized the cells to paclitaxel, but not to carboplatin or cisplatin. AS602801 chemosensitized ovarian CSCs to paclitaxel by reducing the expression of MDR1.

A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity

SUMMARYThe tripeptide glutathione suppresses the iron-dependent, non-apoptotic cell death process of ferroptosis. How glutathione abundance is regulated in the cell and how this regulation alters ferroptosis sensitivity is poorly understood. Using genome-wide human haploid genetic screening technology coupled to fluorescence-activated cell sorting (FACS), we directly identify genes that regulate intracellular glutathione abundance and characterize their role in ferroptosis regulation. Disruption of the ATP binding cassette (ABC)-family transporter multidrug resistance protein 1 (MRP1) prevents glutathione efflux from the cell and strongly inhibits ferroptosis. High levels of MRP1 expression decrease sensitivity to certain pro-apoptotic chemotherapeutic drugs, while collaterally sensitizing to all tested pro-ferroptotic agents. By contrast, disruption of KEAP1 and NAA38, leading to the stabilization of the transcription factor NRF2, increases glutathione levels but only weakly protects from ferroptosis. This is due in part to concomitant NRF2-mediated upregulation of MRP1. These results pinpoint glutathione efflux as an unanticipated regulator of ferroptosis sensitivity.

MIX2: A Novel Natural Multi-Component Modulator of Multidrug-Resistance and Hallmarks of Cancer Cells

Multidrug resistance is one of the key barriers suppressing the effectiveness of drug therapies of malignant tumors. Here, we report a study on the effect of a mix of natural extracts (MIX2) prepared from fresh fruits of Prunus spinosa, Crataegus monogyna, Sorbus aucuparia, and Euonymus europaeus on the classic hallmarks of cancer cells and the expression of multidrug resistance proteins. In the studies, HeLa and T98G cell lines, and classic methods of molecular biology, including RT-qPCR, Western blot, flow cytometry, and confocal imaging, were used. Additionally, migration, adhesion, and proliferation assays were performed. The obtained results indicate that the MIX2 cocktail presents strong anti-cancer properties. MIX2 is not toxic, but at the same time significantly alters the migration, proliferation, and adhesion of tumor cells. Furthermore, it was found that cells exposed to the mixture presented a significantly reduced expression level of genes associated with MDR, including ABCB1, which encodes for glycoprotein P. In vitro data showed that MIX2 effectively sensitizes tumor cells to doxorubicin. We postulate that modulation of the multidrug resistance phenotype of tumors with the use of MIX2 may be considered as a safe and applicable tool in sustaining drug delivery therapies of malignancies.

Belinostat, at Its Clinically Relevant Concentrations, Inhibits Rifampicin-Induced CYP3A4 and MDR1 Gene Expression.

Activation of human pregnane X receptor (hPXR) has been associated with induction of chemoresistance. It has been proposed that such chemoresistance via cytochrome P450/drug transporters can be reversed with the use of antagonists that specifically abrogate agonist-mediated hPXR activation. Unfortunately, proposed antagonists lack the specificity and appropriate pharmacological characteristics that allow these features to be active in the clinic. We propose that, ideally, an hPXR antagonist would be a cancer drug itself that is part of a "cancer drug cocktail" and effective as an hPXR antagonist at therapeutic concentrations. Belinostat (BEL), a histone deacetylase inhibitor approved for the treatment of relapsed/refractory peripheral T-cell lymphoma, and often used in combination with chemotherapy, is an attractive candidate based on its hPXR ligand-like features. We sought to determine whether these features of BEL might allow it to behave as an antagonist in combination chemotherapy regimens that include hPXR activators. BEL represses agonist-activated hPXR target gene expression at its therapeutic concentrations in human primary hepatocytes and LS174T human colon cancer cells. BEL repressed rifampicin-induced gene expression of CYP3A4 and multidrug resistance protein 1, as well as their respective protein activities. BEL decreased rifampicin-induced resistance to SN-38, the active metabolite of irinotecan, in LS174T cells. This finding indicates that BEL could suppress hPXR agonist-induced chemoresistance. BEL attenuated the agonist-induced steroid receptor coactivator-1 interaction with hPXR, and, together with molecular docking studies, the study suggests that BEL directly interacts with multiple sites on hPXR. Taken together, our results suggest that BEL, at its clinically relevant therapeutic concentration, can antagonize hPXR agonist-induced gene expression and chemoresistance.

Resveratrol potentially increased the tumoricidal effect of doxorubicin on SKOV3 cancer stem cells in vitro.

Ovarian cancer is associated with a high percentage of recurrence of tumor and resistance to chemotherapy. Cancer stem cells (CSCs) form a rare population with a significant capacity to begin and expand malignant diseases. Eliminating the drug resistance of CSCs by factors that have fewer side effects to the patient is vital. To investigate the effect of resveratrol (RES) and doxorubicin (DOX) on drug resistance and apoptosis of CSCs; at the first, isolation of CSCs from SKOV3 ovarian carcinoma cells and their dosage adjustment (IC50 ) with RES and DOX was performed. Then, isolated CSCs were treated with RES and DOX IC 50 of 55 and 250 nM, respectively. Subsequently, their effects on drug resistance and cell death were evaluated using real-time polymerase chain reaction, rhodamine 123 uptakes. The results of the present study demonstrated that treatment of SKOV3 with 55 μM of RES and 250 nM of DOX simultaneously increased cell viability in CSCs to DOX after 24 and 48 hours by increasing the expression of Bcl-2-associated X protein (BAX) and caspase-3 genes, and decreased the expression and function of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1) genes indicated by intracellular the rhodamine 123 content. Treatment of RES could increase the activity of DOX cell viability in CSCs originated from SKOV3 ovarian carcinoma and decrease drug resistance capacity to DOX.

Attenuation of STAT3 Phosphorylation Promotes Apoptosis and Chemosensitivity in Human Osteosarcoma Induced by Raddeanin A.

Osteosarcoma (OS) is the most common primary bone malignancy in adolescents. One major obstacle for current OS treatment is drug-resistance. Raddeanin A (RA), an oleanane-type triterpenoid saponin, exerts anti-tumor effects in several tumor models, but the effect of RA in human drug-resistant OS remained to be elucidated. In the present study, we investigated the anti-tumor effects of RA in both drug-sensitive and drug-resistant OS cells and its underlying mechanism. RA inhibited cell proliferation and colony formation and induced apoptotic cell death in a dose-dependent manner in both drug-sensitive and drug-resistant cells. Moreover, RA exposure resulted in the inhibition of interleukin-6 (IL-6)-induced JAK2/STAT3 signaling pathway activation and target gene expression in both drug-sensitive and drug-resistant cells. Meanwhile, we observed significantly increased MDR1 and STAT3 expression in drug-resistant OS cells compared with parental cells. STAT3 overexpression promoted chemo-resistance and MDR1 protein expression in both drug-sensitive OS cells and drug-resistant OS cells, while inhibiting STAT3 with siRNA sensitized OS cells to doxorubicin treatment. In addition, RA synergistically increased doxorubicin toxicity by increasing its cellular uptake, ablating efflux and downregulating MDR1 in drug-resistant cells with attenuation of STAT3 Phosphorylation. Finally, RA suppressed in vivo tumor growth and induced apoptosis in nude mouse using drug-resistant OS tibia orthotopic model. Taken together, RA is a promising potential therapeutic for the treatment of doxorubicin resistance in OS.

Coexistence of (CCND1/PRAD-1/BCL1) juxtaposition and drug resistance gene is a marker for vincristine-resistant B-non-Hodgkin’s lymphoma

Background CCND1/PRAD-1/BCL1 juxtaposition activates immunoglobulin heavy chain (IgH) enhancing effect on cyclin D1 expression. This leads to overexpression of cyclin D1, a cell-cycle regulator. Cyclin D1 can function as an oncogene growth advantage for tumor cells by way of cell-cycle progression. Multidrug resistance (MDR)1 gene expression leads to overproduction of P-glycoprotein (P-gp) which leads to chemotherapy resistance causing poor prognosis and short survival [overall survival (OS) and disease free survival(DFS)] in B-non-Hodgkin’s lymphoma (NHL). Objective To evaluate BCL1/IgH juxtaposition by fluorescent in-situ hybridization (FISH) and MDR1 protein expression by immunohistochemistry in B-non Hodgkin lymphoma (B-NHL) and to clarify if these genes have some relevance to developing vincristine treatment resistance of B-NHL and their correlation with clinical outcome. Patients and methods Fifty-three bone marrow biopsy samples with B-NHL were included in the study for the expression of t(11;14) BCL1/IgH in various B-cell lymphomas by FISH and MDR1/P-gp by immunohistochemistry and its relation to the outcome and chemotherapy resistance. Results In this study t(11;14) detected by FISH was positive in 64.2%. It was positive in (66.7%) of mantle cell lymphoma, in 64.3% of small cell lymphoma, and in 60% of large cell lymphoma. The detection frequency of MDR1/P-gp by immunohistochemistry in mantle cell lymphoma, small cell lymphoma and large cell lymphoma, is 80, 82, and 80%, respectively. Conclusion In this study, the frequency of t(11;14) detection by FISH and MDR1/P-gp expression by immunohistochemistry in Egyptian patients were consistent with other studies and the coexistence of MDR1/P-gp and t(11;14) in B-NHL is important before treatment to allow the design of novel drug regimens.

Gancao (Glycyrrhizae Radix) provides the main contribution to Shaoyao-Gancao decoction on enhancements of CYP3A4 and MDR1 expression via pregnane X receptor pathway in vitro

BackgroundChinese herbal formula Shaoyao Gancao decoction (SGD) is often used as an adjuvant with chemotherapeutic agents to treat cancer. Due to the herb-drug interactions, the alternations of drug metabolic enzyme and drug transporters induced by SGD deserve to be explored. We aimed to investigate the effect of SGD on the pregnane X receptor (PXR)-mediated transcriptional regulation of cytochrome P450 3A4 (CYP3A4) and drug transporter multidrug resistance protein 1 (MDR1) in vitro. Besides, we assessed the contribution of constituent herbs to SGD on the regulation of CYP3A4 and MDR1.MethodsThe dual luciferase reporter gene system containing the hPXR expression plasmid and the reporter gene plasmid of CYP3A4 or MDR1 was co-transfected to HepG2 and Caco2 cells. Luciferase activities were determined using a Dual-luciferase reporter assay kit. The gene expression of CYP3A4 and MDR1 in the hPXR-transfected LS174T cells were assessed by real-time qPCR. Finally, the contribution of constituent herbs from SGD was evaluated.ResultsSGD, Shaoyao and Gancao concentration-dependently increased promoter activities of CYP3A4 and MDR1 in vitro. Moreover, SGD, Shaoyao and Gancao up-regulated CYP3A4 and MDR1 mRNA in hPXR-transfected LS174T cells. As the herbal constituent of SGD, Gancao possesses significantly higher levels of metabolic enzyme and drug transporters compared with Shaoyao.ConclusionSGD tends to enhance CYP3A4 and MDR1 expression via PXR pathway, especially Gancao provides the main contribution. This study highlights a potential in vitro mechanism for SGD on the regulation of drug metabolic enzymes and drug transporters.

LevistolideA synergistically enhances doxorubicin‑induced apoptosis of k562/dox cells by decreasing MDR1 expression through the ubiquitin pathway.

Multidrug resistance (MDR) is one of the main reasons underlying failure of cancer chemotherapy. Certain natural compounds may help prevent MDR, and may be used in combination with chemotherapeutic agents to enhance their efficacy. Levistolide A is a natural product that is extracted from the rhizome of Angelicae sinensis (Oliv.), which has been used as an essential component of antitumor formulas since ancient times in China. The present study conducted the following experiments: MTT assay, apoptosis analysis, cellular doxorubicin accumulation assay, immunoblotting and reverse transcription‑quantitative polymerase chain reaction, to investigate whether levistolide A enhance doxorubicin‑induced apoptosis of k562/dox cells and to determine the molecular mechanisms involved. When combined with doxorubicin, levistolide A exhibited a synergistic effect and induced cytotoxicity in k562/dox cells. Drug accumulation studies revealed that levistolide A increased the intracellular concentration of doxorubicin in a dose‑dependent manner. Cell apoptosis experiments indicated that levistolide A increased the sensitivity of k562/dox cells to doxorubicin. Furthermore, detection of reactive oxygen species (ROS) revealed that levistolide A enhanced doxorubicin‑induced cell death by increasing the levels of ROS. Mitochondrial potential detection with JC‑1 staining also indicated that levistolide A synergistically enhanced doxorubicin‑induced cell death. Immunoblotting demonstrated that levistolide A enhanced doxorubicin‑induced cell death by decreasing the expression levels of B‑cell lymphoma 2 and increasing caspase 3 expression. Furthermore, multidrug resistance protein 1 (MDR1) expression in k562/dox cells was downregulated by levistolide A in a dose‑dependent manner, thus suggesting that levistolide A may modulate MDR1 during cancer therapy. Therefore, the combination of levistolide A with doxorubicin could result in more effective and less toxic anticancer regimens.

Multi-drug resistance protein 2 (MRP2) expression, adjuvant tamoxifen therapy, and risk of breast cancer recurrence: a Danish population-based nested case-control study

Background: Adjuvant tamoxifen therapy approximately halves the risk of recurrence in estrogen receptor-positive (ER+) breast cancer patients, but many women respond insufficiently to therapy. Expression of multi-drug resistance protein 2 (MRP2) in breast cancer may potentiate tamoxifen resistance. Thus, we investigated the expression of MRP2 in breast cancer as a predictor of tamoxifen therapy effectiveness.Material and methods: We conducted a case-control study nested in the Danish Breast Cancer Group clinical database. The study included women aged 35–69 years diagnosed with stage l–lll breast cancer during 1985–2001, in Jutland, Denmark. We identified 541 recurrent breast cancers (cases) among women with estrogen receptor positive (ER+) disease treated with tamoxifen for at least 1 year (ER+/TAM+) and 300 cases among women with estrogen receptor-negative (ER−) disease, never treated with tamoxifen (ER−/TAM−). We matched one recurrence-free control to each recurrent case. We retrieved paraffin-embedded primary tumor tissue for all patients, and all available recurrent tumor tissue from pathology archives. MRP2 expression was evaluated using immunohistochemistry. We computed odds ratios (ORs) and 95% confidence intervals (95% CIs) associating MRP2 expression (positive vs. none) with breast cancer recurrence in conditional logistic regression models. We compared MRP2 expression in paired primary- and recurrent tumors.Results: MRP2 expression was more prevalent in the ER+/TAM + group, than in the ER−/TAM − group. No predictive utility of MRP2 for breast cancer recurrence was found in the ER+/TAM + group (ORadj = 0.96, 95% CI 0.70, 1.33). Further, no prognostic utility was found in the ER−/TAM − group (ORadj = 0.81, 95% CI 0.53, 1.23). MRP2 expression was not increased in recurrent versus primary tumors.Conclusions: MRP2 expression is neither a predictive marker of tamoxifen effectiveness nor a prognostic marker in breast cancer.

Ovarian hormones modulate multidrug resistance transporters in the ovary

BackgroundMultidrug resistance transporters (MDRs) are transmembrane proteins that efflux metabolites and xenobiotics. They are highly conserved in sequence and function in bacteria and eukaryotes and play important roles in cellular homeostasis, as well as in avoidance of antibiotics and cancer therapies. Recent evidence also documents a critical role in reproductive health and in protecting the ovary from environmental toxicant effects. The most well understood MDRs are MDR-1 (P-glycoprotein (P-gp) also known as ABCB1) and BCRP (breast cancer resistance protein) and are both expressed in the ovary. We have previously shown that MDR-1 mRNA steady state expression changes throughout the murine estrous cycle, but expression appears to increase in association with the surge in estradiol during proestrus.MethodsHere we test the model that MDR-1 and BCRP are regulated by estrogen, the major hormonal product of the ovary. This was performed by administering 6-week-old female mice either sesame oil (vehicle control) or oral ethinyl estradiol at 1 μg, 10 μg, and 100 μg or PROGESTERONE at 0.25mg, 0.5 mg or 1 mg or a combination of both for 5 days. The mice were then sacrificed, and the ovaries were removed and cleaned. Ovaries were used for qPCR, immunoblotting, and immnunolabeling.ResultsWe found that oral ethinyl estradiol did not influence the steady state mRNA of MDR-1 or BCRP. Remarkably, the effect on mRNA levels neither increased or decreased in abundance upon estrogen exposures. Conversely, we observed less MDR-1 protein expression in the groups treated with 1 μg and 10 μg, but not 100 μg of ethinyl estradiol compared to controls. MDR-1 and BCRP are both expressed in pre-ovulatory follicles. When we tested progesterone, we found that MDR-1 mRNA increased at the dosages of 0.25 mg and 0.5 mg, but protein expression levels were not statistically significant. Combined oral ethinyl estradiol and progesterone significantly lowered both MDR-1 mRNA and protein.ConclusionsProgesterone appears to influence MDR-1 transcript levels, or steady state levels. This could have implications for better understanding how MDR-1 can be modulated during times of toxic exposure. Understanding the normal physiology of MDR-1 in the ovary will expand the current knowledge in cancer biology and reproduction.

Association between NF-κB expression and drug resistance of liver cancer.

Association between the expression of nuclear factor κB (NF-κB) and the drug resistance of hepatoma cells was investigated. HepG-2 cells and HepG2/ADM cells were cultured, respectively. The morphology and status of the two groups of cells were observed by cell white light. The immunofluorescence by NF-κB and MDR1 staining on HepG-2 cells and HepG2/ADM cells, respectively, was applied and the fluorescence expression in the two groups of cells was observed. RT-qPCR was used to detect the expression of NF-κB and MDR1 mRNA, the NF-κB and MDR1 protein expression was detected by western blot analysis. The results of cell white illumination showed that the structure of HepG-2 and HepG2/ADM cells was complete and the cell morphology was normal, and there was no significant difference, and could be used for comparative study. Immunofluorescence staining showed that the expression of NF-κB and MDR1 in HepG-2 cells was very low, while the expression of NF-κB and MDR1 in HepG2/ADM cells was increased significantly. The RT-qPCR results showed that NF-κB and MDR1 mRNA expression in HepG-2 cells was very low, while NF-κB and MDR1 mRNA expression in HepG-2/ADM cells was significantly increased, and western blot results showed that NF-κB and MDR1 protein expression in HepG-2 cells was very low, while NF-κB and MDR1 protein expression in HepG-2/ADM cells was increased significantly. The results of variance analysis showed that there was significant difference in the expression of the control group and paeonol group (P<0.01). In conclusion, the expression of NF-κB in the drug-resistant cells of liver cancer is closely related to the resistance-related gene MDR1. This result may provide a new solution for the drug resistance of liver cancer.

Expression of multidrug-resistance associated proteins in human retinoblastoma treated by primary enucleation.

To reveal the expression of multidrug-resistance associated proteins: glutathione-S-transferase π (GSTπ), P-glycoprotein (P-gp) and vault protein lung resistance protein (LRP) in retinoblastoma (RB) without any conservative treatment before primary enucleation and to correlate this expression with histopathological tumor features. A total of 42 specimens of RB undergone primary enucleation were selected for the research. Sections from the formalin-fixed, paraffin-embedded specimens were stained with HE and immunohistochemistry to detect the expression of GSTπ, P-gp and LRP. GSTπ was expressed in 39/42 (92.86%) RBs and in 9/9 (100%) well-differentiated RBs. P-gp/GSTπ was found in 30 (71.42%) of 42 RBs. Totally 9 (21.43%) tumors were well differentiated and 33 (78.57%) were poorly differentiated. Totally 15 (35.71%) eyes had optic nerve (ON) tumor invasion, 36 (85.71%) had choroidal tumor invasion, and 14 (33.33%) had simultaneous choroidal and ON invasion. There was no statistically significant relationship between P-gp, GSTπ, LRP positivity and the degree of ocular layer tumor invasion and ON tumor invasion (P>0.05). RB intrinsically expresses GSTπ, P-gp and LRP. GSTπ expression is positive in 100% well-differentiation ones, so in which way it is correlated with differentiation. But the other two proteins expressions are not related to tumor differentiation and to the degree of tumor invasion. GSTπ may be a new target of chemotherapy in RB.

Silencing microRNA-16 induces drug resistance of hepatoma cell lines via nuclear factor-κB pathway

Objective Study on the mechanism of sliencing microRNA (miRNA, miR)-16 on chemoresistance in SMMC-7721. Methods The lentiviral vectors were used to construct stable cell lines. To determine whether constructed successfully, the expression of miR-16 was detected by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and inhibitor of kappaB kinase beta (IKBKB), the target gene of miR-16, was detected by Western blotting. The sensitivity and resistance mechanism of the stable cell lines to paclitaxel were detected by Western blotting and thiazole blue (MTT). Results The expression of miR-16 in 7721-miR16 group was significantly higher than SMMC-7721 and 7721-mock groups (4.63±1.32 vs. 1.00±0.30 vs. 0.93±0.37, P =0.000). The expression of IKBKB in 7721-shmiR16 was 25.73 times than the controls (P=0.000). The expression of multidrug resistance protein 1 (MDR1) in 7721-shmiR16 was increased and the half maximal inhibitory concentration (IC50) to paclitaxel was 4.903 μmol/L, 10.64 times than the control group (0.461 μmol/L) (P=0.000). The results of western showed that silencing miR-16 induced resistance mainly through activating nuclear factor-kappaB (NF-κB) pathway. Conclusion Silencing miR-16 in SMMC-7721 significantly increases IKBKB expression leadind paclitaxel resistance. Key words: Hepatoma; MicroRNA-16; Paclitaxel; Drug resistance; Inhibitor of kappaB kinase beta

Appendix-derived Pseudomyxoma Peritonei (PMP): Molecular Profiling Toward Treatment of a Rare Malignancy.

Pseudomyxoma peritonei (PMP) is a rare malignancy originating from the appendix, characterized by disseminated mucinous tumor implants on peritoneal surfaces. We examined the role of multiplatform molecular profiling to study biomarker-guided treatment strategies for this rare malignancy. A total of 54 patients with appendix-derived PMP were included in the study. Tests included one or more of the following: gene sequencing (Sanger or next generation sequencing), protein expression (immunohistochemistry), and gene amplification (C/fluorescent in situ hybridization). Targeted sequencing of 47 genes detected variants in KRAS (81%), GNAS (74%), SMAD4 (16%), and ATM (16%). Mutations were found at low frequencies (n=1 to 2) in APC, BRAF, PIK3CA, MLH1, and TP53. GNAS and KRAS co-occurrence was found in 87%. Protein overexpression was found in epidermal growth factor receptor (83%), cyclooxygenase-2 (73%), cMET (63%), cKIT (58%), and platelet-derived growth factor receptor alpha (58%). Immune checkpoint expression was found in 36% (programmed cell death protein 1) and 18% (programmed death-ligand 1). Surrogate markers of cell proliferation were found at low rates (TLE3 23%, TOP2A 22%), consistent with the slow-growing biology of PMP. Phosophatase and tensin homolog was intact (wild type [100%]) and positive (immunohistochemistry [80%]). Patients exhibited stable microsatellite status and mismatch repair proficiency (93%). Importantly, multidrug resistance protein expression was elevated (100% BCRP, 94% MRP1, 88% PGP). Markers for gemcitabine (RRM1), fluorouracil (TS), oxaliplatin (ERCC1), and irinotecan (TOPO1) chemosensitivities were detected at favorable rates: 93%, 87%, 77% and 65%, respectively. Molecular profiling by multiple platforms identified potential therapies for the nontargetable KRAS-mutated population. The role of cMET-targeted therapeutics and immune checkpoint inhibitors merits further investigation. Biomarker-guided selection of cytotoxic chemotherapies may facilitate efficacy to systemic treatment.