ABSTRACT
Drug resistance in cells is a major impedance to successful treatment of lung cancer. Taxus chinensis var. inhibits the growth of tumor cells and promotes the synthesis of interleukins 1 and 2 and tumor necrosis factor, enhancing immune function. In this study, T. chinensis var.-induced cell death was analyzed in lung cancer cells (H460) enriched for stem cell growth in a defined serum-free medium. Taxus-treated stem cells were also analyzed for Rhodamine 123 (Rh-123) expression by flow cytometry, and used as a standard functional indicator of MDR. The molecular basis of T. chinensis var.-mediated drug resistance was established by real-time PCR analysis of ABCC1, ABCB1, and lung resistance-related protein (LRP) mRNA, and western blot analysis of MRP1, MDR1, and LRP. Our results revealed that stem cells treated with higher doses of T. chinensis var. showed significantly lower growth inhibition rates than did H460 cells (P < 0.05). The growth of stem and H460 cells treated with a combination of T. chinensis var. and cisplatin was also significantly inhibited (P < 0.05). Rh-123 was significantly accumulated in the intracellular region and showed delayed efflux in stem cells treated with T. chinensis var. (P < 0.05), compared to those treated with verapamil. T. chinensis var.-treated stem cells showed significant downregulation of the ABCC1, ABCB1, and LRP mRNA and MRP1, MDR1, and LRP (P < 0.05) compared to H460 cells. Thus, T. chinensis var.-mediated downregulation of MRP1, MDR1, and LRP might contribute to the reversal of drug resistance in non-small cell lung cancer stem cells.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic , Neoplastic Stem Cells/drug effects , Plant Extracts/pharmacology , Taxus/chemistry , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Antineoplastic Agents/chemistry , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cisplatin/pharmacology , Drug Combinations , Drug Resistance, Neoplasm/genetics , Drugs, Chinese Herbal , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Plant Extracts/chemistry , Rhodamine 123/metabolism , Signal Transduction , Vault Ribonucleoprotein Particles/antagonists & inhibitors , Vault Ribonucleoprotein Particles/genetics , Vault Ribonucleoprotein Particles/metabolismABSTRACT
Although two classes of antivirals, NA inhibitors and M2 ion channel blockers, are licensed for influenza treatment, dual resistant mutants, including highly pathogenic H5N1 viruses, have appeared. Alternative treatment options are, therefore, needed. Influenza A viral RNA (vRNA) transcription/replication is a promising target for antiviral development, since it is essential for virus replication. Accordingly, an efficient and reliable method to identify vRNA transcription/replication inhibitors is desirable. Here, we developed a cell-based screening system by establishing a cell line that stably expresses influenza viral ribonucleoprotein complex (vRNP). Compound library screening using this cell line allowed us to identify a compound that inhibits vRNA transcription/replication by using reporter protein expression from virus-like RNA as a readout and virus replication in vitro. vRNP-expressing cells have potential as a simple and convenient high-throughput screening (HTS) system, and, thus, are promising to identify vRNA transcription/replication inhibitors for various RNA viruses, especially for primary screens.
Subject(s)
Antiviral Agents/pharmacology , High-Throughput Screening Assays/methods , Influenza A virus/drug effects , Influenza A virus/physiology , RNA, Viral/drug effects , Virus Replication/drug effects , Animals , Dogs , Drug Evaluation, Preclinical/methods , Genetic Vectors/genetics , HEK293 Cells , Humans , Influenza A virus/genetics , Influenza, Human/drug therapy , Influenza, Human/virology , Madin Darby Canine Kidney Cells , RNA Viruses/drug effects , RNA Viruses/genetics , RNA, Viral/genetics , Vault Ribonucleoprotein Particles/drug effects , Vault Ribonucleoprotein Particles/genetics , Viral Proteins/genetics , Virus Replication/geneticsABSTRACT
Tanshinone IIA is known to induce apoptosis in several types of cancer cells. However, little is known about its activity in chemoresistant cells. The aim of this study was to investigate the anticancer properties of tanshinone IIA in cisplatin-resistant human ovarian cancer COC1/DDP cells in vitro. We used a variety of methods to measure cell viability, the resistance index (RI) of cisplatin, cellular apoptosis, p38 mitogen-activated protein kinase (MAPK) expression and phosphorylation, and the mRNA expression of several genes implicated in drug resistance including survivin, Caspase-3, excision repair cross-complementing gene 1 (ERCC1), multidrug resistance (MDR), lung resistance protein (LRP) and glutathione-S-transferase-π (GST-π). We found that tanshinone IIA time- and dose-dependently inhibited the proliferation of COC1/DDP cells and caused significant apoptosis. Western blotting revealed that tanshinone IIA also increased phospho-p38 MAPK in a time- and dose-dependent manner. After treatment by tanshinone IIA for 48 h, the RI of cisplatin and the mRNA expression of survivin, ERCC1 and LRP were all significantly decreased. Furthermore, blockade of p38 signal transduction decreased apoptotic cell rates and dramatically elevated the mRNA expression of the survivin, ERCC1 and LRP genes. We therefore conclude that tanshinone IIA induces apoptosis and reduces cisplatin resistance in COC1/DDP cells and thus causes significant growth inhibitory effects. This mechanism appears to involve p38-mediated downregulation of survivin, ERCC1 and LRP mRNA expression.
Subject(s)
Abietanes/pharmacology , Apoptosis/drug effects , Carcinoma/pathology , DNA-Binding Proteins/genetics , Endonucleases/genetics , Ovarian Neoplasms/pathology , Vault Ribonucleoprotein Particles/genetics , p38 Mitogen-Activated Protein Kinases/physiology , Abietanes/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Carcinoma/drug therapy , Carcinoma/genetics , Cell Line, Tumor , Cisplatin/therapeutic use , DNA-Binding Proteins/metabolism , Down-Regulation/drug effects , Drug Evaluation, Preclinical , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Endonucleases/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Signal Transduction/drug effects , Vault Ribonucleoprotein Particles/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
OBJECTIVE: To explore the pharmacologic effects of Chinese medicine Bushen Huayu Jiedu Compound Recipe (BSHYJDR) in drug-resistance cells of lung cancer. METHODS: Human lung adenocarcinoma A549/DDP cell strain was selected, serum pharmacology and flow cytometer (FCM) method were adopted, S180 tumor-bearing mice and normal mice were given, through gastrogavage, different doses of a decocted concentration of BSHYJDR. Serum from the abdominal aorta was taken to observe the effect of drug-serum on cisplatin (DDP) concentration, free Ca2+ concentration and the expression of lung drug-resistance protein LRP-56 in A549/DDP cells. RESULTS: Compared with the drug-resistance group, the intracellular DDP concentration in the group taking a high dose and the normal group of Chinese medicine showed significant difference (P<0.05), while no significant difference was found in the low-dose group (P>0.05). Compared with the drug-resistance group, the Ca2+ concentration in cells and the expression of LRP in lung cancer drug-resistance cells A549/DDP of the high-dose group, the low-dose group and the normal group of Chinese medicine were significantly different (all P<0.01), the LRP expression of the normal group was obviously higher than that of the drug-resistance group (P<0.05). CONCLUSION: It was indicated that serum containing Chinese medicine BSHYJDR in the tumor-bearing mice and the normal mice had certainly different, tumor-bearing mice serum containing could improve drug concentration in lung cancer drug-resistance cells, prevent the inflow and release of Ca2+, and inhibit the expression of the drug-resistance gene in the lung cancer drug-resistance cells, which might be the mechanism of BSHYJDR in enhancing the efficacy in reversing and inhibiting tumor.
Subject(s)
Lung Neoplasms/drug therapy , Medicine, Chinese Traditional , Animals , Calcium/metabolism , Cell Line, Tumor , Cisplatin/pharmacology , Drug Resistance, Neoplasm , Humans , Lung Neoplasms/pathology , Mice , Vault Ribonucleoprotein Particles/geneticsABSTRACT
PURPOSE: 5-Fluorouracil failure and drug resistance, which often occurs during chemotherapy, is still a great obstacle to the success of human colon cancer treatment. Thus, the comparative study of markers of drug resistance in cancer cells before and after chemotherapy may be extremely helpful in the selection of the appropriate chemotherapeutic drug in colon cancer patients who fail adjuvant treatment with 5-fluorouracil. In the present study we examined the differential expression of three multidrug resistance-related proteins (i.e., P-glycoprotein, MRP, and LRP) and of topoisomerase IIalpha in a series of 20 primary colon carcinomas and their recurrences. METHODS: All markers were determined at tissue level by three-step immunohistochemistry using appropriate monoclonal antibodies, and the markers' immunopositivity was quantified by image analysis. In addition, Feulgen stain was used for the assessment of nuclear DNA content of malignant cells at their primary location. RESULTS: Some degree of aneuploidy was detected in all primary carcinomas. The immunoexpression of the three multidrug resistance-related proteins did not change significantly, either qualitatively (positivity vs negativity) or quantitatively, after chemotherapy. On the contrary, the percentages of topoisomerase IIalpha-positive malignant cells were significantly increased in the tumour recurrences by comparison to their primary locations ( P=0.011). CONCLUSIONS: According to our results, increased topoisomerase IIalpha immunohistochemical expression appears to be part of the malignant cells' phenotype in recurrent colon cancers. Therapeutic options after failure of 5-fluorouracil-based treatment could therefore include appropriate topoisomerase IIalpha-targeted drugs.