Bcl-2 and Bcl-xL are important for the induction of paclitaxel resistance in human hepatocellular carcinoma cells

In this study we have investigated the mechanism underlying resistance to the chemotherapeutic drug paclitaxel in tumors of hepatocellular carcinoma (HCC) patients. Treatment with paclitaxel led to potent inhibition of growth of Hep3B hepatoma cells, but did not affect the growth properties of SNU-368 and SNU-398 cell lines that were established from primary HCC tumors. The growth inhibitory effect induced by paclitaxel correlated with levels of intracellular p21 and resulted in cell cycle arrest at the G2/M phase. However, paclitaxel treatment did not alter intracellular p53 levels. Instead, SNU-398 cells express high levels of the anti-apoptotic Bcl-2 and Bcl-x(L) proteins and the level of Bcl-x(L) could be further induced upon paclitaxel treatment. In contrast, Hep3B cells express pro-apoptotic members of the Bcl family and fail to induce Bcl-x(L) upon paclitaxel treatment. Therefore, these results strongly suggest that Bcl-2 and Bcl-x(L) play an important role in mediating resistance to paclitaxel.     

Resistance to paclitaxel in hepatoma cells is related to static JNK activation and prohibition into entry of mitosis

Hepatocellular carcinoma (HCC) generally shows chemoresistant features to anticancer agents. Paclitaxel has been clinically used in the treatment of various cancers. However, effect of paclitaxel on HCC has not been adequately addressed. Here, we found two categories of hepatoma cells in response to paclitaxel. Paclitaxel effectively decreased the cell viability of SNU475, Hep3B, and SNU387 HCC cells and Chang liver cells (death prone). In contrast, the other five hepatoma cell lines (SNU449, SNU398, SUN368, SNU354, and HepG2 cells) were resistant to paclitaxel (death reluctant). In response to paclitaxel, Bcl-2 was highly phosphorylated in death-prone cells, whereas much less Bcl-2 was phosphorylated in death-reluctant cells. Cotreatment with SP600125, an inhibitor JNK, significantly reduced the phosphorylated Bcl-2 in death-prone cells and caused a significant reduction in cell death. The reduced cell death was due to prohibition into mitotic entry as evidenced by low cyclin B(1)/Cdk1 kinase activity. In death-reluctant cells, inbuild-phospho-JNK levels were high but no longer activated in response to paclitaxel. We found that paclitaxel combined with caffeine or UCN-01, inhibitors of G(2) DNA damage checkpoint, was able to partially overcome resistance to paclitaxel in these cells. Thus our data provide the molecular basis of paclitaxel resistance in hepatoma cells, and appropriate combination therapy may increase treatment efficacy.     

Caveolin-1 interacts with a lipid raft-associated population of fatty acid synthase

Fatty Acid Synthase (FASN), a cytoplasmic biosynthetic enzyme, is the major source of long-chain fatty acids, particularly palmitate. Caveolin-1 (Cav-1) is a palmitoylated lipid raft protein that plays a key role in signal transduction and cholesterol transport. Both proteins have been implicated in prostate cancer (PCa) progression, and Cav-1 regulates FASN expression in a mouse model of aggressive PCa. We demonstrate that FASN and Cav-1 are coordinately up-regulated in human prostate tumors in a hormone-insensitive manner. Levels of FASN and Cav-1 protein expression discriminated between localized and metastatic cancers, and the two proteins exhibited analogous subcellular locations in a tumor subset. Endogenous FASN and Cav-1 were reciprocally co-immunoprecipitated from human and murine PCa cells, indicating that FASN forms a complex with Cav-1. FASN, a cytoplasmic enzyme, was induced to associate transiently with lipid raft membranes following alterations in signal transduction within the Src, Akt and EGFR pathways, suggesting that co-localization of FASN and Cav-1 is dependent on activation of upstream signaling mediators. A Cav-1 palmitoylation mutant, Cav-1C133/143/156S, that prevents phosphorylation by Src, did not interact with FASN. When overexpressed in Cav-1-negative PCa cells, Cav-1C133/143/156S caused a reduction of both Src and Akt levels, as well as of their active, phosphorylated forms, in comparison with wild type Cav-1. These findings suggest that FASN and Cav-1 physically and functionally interact in PCa cells. They also imply that palmitoylation within this complex is involved in tumor growth and survival.     

Caveolin-1 Is Up-Regulated by GLI1 and Contributes to GLI1-Driven EMT in Hepatocellular Carcinoma

Caveolin-1 (Cav-1) has been recently identified to be over-expressed in hepatocellular carcinoma (HCC) and promote HCC cell motility and invasion ability via inducing epithelial-mesenchymal transition (EMT). However, the mechanism of aberrant overexpression of Cav-1 remains vague. Here, we observed that Cav-1 expression was positively associated with GLI1 expression in HCC tissues. Forced expression of GLI1 up-regulated Cav-1 in Huh7 cells, while knockdown of GLI1 decreased expression of Cav-1 in SNU449 cells. Additionally, silencing Cav-1 abolished GLI1-induced EMT of Huh7 cells. The correlation between GLI1 and Cav-1 was confirmed in tumor specimens from HCC patients and Cav-1 was found to be associated with poor prognosis after hepatic resection. The relationship between protein expression of GLI1 and Cav-1 was also established in HCC xenografts of nude mice. These results suggest that GLI1 may be attributed to Cav-1 up-regulation which plays an important role in GLI1-driven EMT phenotype in HCC.     

Chloride channel-3 mediates multidrug resistance of cancer by upregulating P-glycoprotein expression

Chloride channel-3 (ClC-3), a member of the ClC family of voltage-gated Cl⁻ channels, is involved in the resistance of tumor cells to chemotherapeutic drugs. Here, we report a new mechanism for ClC-3 in mediating multidrug resistance (MDR). ClC-3 was highly expressed in the P-glycoprotein (P-gp)-dependent human lung adenocarcinoma cell line (A549)/paclitaxel (PTX) and the human breast carcinoma cell line (MCF-7)/doxorubicin (DOX) resistant cells. Changes in the ClC-3 expression resulted in the development of drug resistance in formerly drug-sensitive A549 or MCF-7 cells, and drug sensitivity in formerly drug-resistant A549/Taxol and MCF-7/DOX cells. Double transgenic MMTV-PyMT/CLCN3 mice with spontaneous mammary cancer and ClC-3 overexpression demonstrated drug resistance to PTX and DOX. ClC-3 expression upregulated the expression of MDR1 messenger RNA and P-gp by activating the nuclear factor-κB (NF-κB)-signaling pathway. These data suggest that ClC-3 expression in cancer cells induces MDR by upregulating NF-κB-signaling-dependent P-gp expression involving another new mechanism for ClC-3 in the development of drug resistance of cancers.     

Co-expression of fatty acid synthase and caveolin-1 in pancreatic ductal adenocarcinoma: Implications for tumor progression and clinical outcome

Pancreatic cancer is a devastating disease that afflicts over 35,000 Americans every year. Since therapeutic options are limited, understanding the molecular aspects of this disease is critical for moving towards targeted treatment of this aggressive form of cancer. Caveolin-1 (Cav-1) and fatty acid synthase (FASN) are two proteins that have been shown to be dysregulated in a number of cancers. Functionally these proteins have been shown to be involved in the process of tumorigenesis. We thus surveyed the expression of both these critical proteins in a series of pancreatic precancerous lesions (pancreatic intraepithelial neoplasia, PanINs) and pancreatic cancers. Cav-1 and FASN expression correlated predominantly with clinical characteristics, such as histologic grade and advanced tumor stage (e.g. high Cav-1 and FASN expression correlated with poor differentiation status) and a significant survival advantage was found in patients with low co-expressing FASN and Cav-1 tumors. Cav-1 and FASN expression was absent in PanIN lesions and the normal ducts and acini. Of note, Cav-1 expression was detected in the fibroblasts of the desmoplastic pancreatic cancer stroma, but not in stromal cells of the normal pancreas. Mechanistically, these data support the notion that these proteins are co-regulated either directly or indirectly by another factor. Importantly, the co-expression of these proteins significantly correlates with clinical features and survival status of pancreatic cancer patients. Thus, Cav-1 and FASN may functionally cooperate in the process of pancreatic tumorigenesis, and as such, may be good candidate prognostic markers and targets for therapeutic intervention.     

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.     

Vitamin E protects DNA from oxidative damage in human hepatocellular carcinoma cell lines

Expression of multiple drug resistant (MDR) phenotype and over-expression of P-glycoprotein (P-gp) in the human hepatocellular carcinoma (HCC) cell clone P1(0.5), derived from the PLC/PRF/5 cell line (P5), are associated with strong resistance to oxidative stress and a significant (p < 0.01) increase in intracellular vitamin E content as compared with the parental cell line. This study evaluates the role of vitamin E in conferring resistance to drugs and oxidative stress in P1(0.5) cells. Parental drug-sensitive cells, P5, were incubated in alpha-tocopherol succinate (alpha-TS, 5 microM for 24 h) enriched medium to increase intracellular vitamin E content to levels comparable to those observed in P1(0.5) cells at basal conditions. Susceptibility to lipid peroxidation and oxidative DNA damage were assessed by measuring the concentration of thiobarbituric-reactive substances (TBARS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) at basal and after experimental conditions. Cell capacity to form colonies and resistance to doxorubicin were also studied. P5 cells, treated with alpha-TS, became resistant to ADP-Fe3+ and to ionizing radiation-induced lipid peroxidation as P1(0.5) cells. Exposure to ADP-Fe3+ or ionizing radiation increased TBARS and the 8-OHdG content in the P5 cells, while vitamin E enrichment abolished these effects. Irradiation doses at 5 cGy increased TBARS and 8-OHdG. They also inhibited cell capacity to form colonies in the untreated P5 cells. Incubation with alpha-TS fully reverted this effect and significantly (p < 0.01) reduced the inhibitory effect of cell proliferation induced by irradiation doses at >500 cGy. Resistance to doxorubicin was not affected by alpha-TS. These observations demonstrate the role of vitamin E in conferring protection from lipid peroxidation, ionizing radiation and oxidative DNA damage on the human HCC cell line. They also rule out any role of P-gp over-expression as being responsible for these observations in cells with MDR phenotype expression.     

A New 2α,5α,10β,14β-tetraacetoxy-4(20),11-taxadiene (SIA) Derivative Overcomes Paclitaxel Resistance by Inhibiting MAPK Signaling and Increasing Paclitaxel Accumulation in Breast Cancer Cells

Tumor resistance due to multiple mechanisms seriously hinders the efficacy of chemotherapy drugs such as paclitaxel. The most widely studied P-glycoprotein inhibitors still have limited ability to reverse resistance in the clinic. In this study, NPB304, a novel Sinenxan A (SIA) derivative, was found to significantly sensitize resistant breast cancer cells to paclitaxel in vitro and in vivo. Treatment with NPB304 increased paclitaxel-induced apoptosis in a p53-dependent manner through PARP cleavage. Importantly, NPB304 enhanced the antitumor effect of paclitaxel in resistant breast tumor xenografts in nude mice without significantly affecting weight loss. NPB304 regulated cell resistance through inhibition of MAPK pathway components, including p-ERK and p-p38. Moreover, NPB304 increased paclitaxel accumulation by affecting P-gp function. In addition to increasing Rhodamine 123 accumulation, NPB304 promoted bidirectional permeability but decreased the efflux ratio of paclitaxel in a Caco-2 monolayer model, thereby increasing the intracellular concentration of paclitaxel. Similarly, NPB304 increased the concentration of paclitaxel in the resistant tumor tissue. Hence, NPB304 is a novel compound that promotes the sensitization of resistant cells to paclitaxel through multiple mechanisms and has the potential for use in combination therapies to treat resistant breast cancer.     

Over-expression of cytochrome P450 CYP6CM1 is associated with high resistance to imidacloprid in the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae)

The two most damaging biotypes of Bemisia tabaci, B and Q, have both evolved strong resistance to the neonicotinoid insecticide imidacloprid. The major mechanism in all samples investigated so far appeared to be enhanced detoxification by cytochrome P450s monooxygenases (P450s). In this study, a polymerase chain reaction (PCR) technology using degenerate primers based on conserved P450 helix I and heme-binding regions was employed to identify P450 cDNA sequences in B. tabaci that might be involved in imidacloprid resistance. Eleven distinct P450 cDNA sequences were isolated and classified as members of the CYP4 or CYP6 families. The mRNA expression levels of all 11 genes were compared by real-time quantitative RT-PCR across nine B and Q field-derived strains of B. tabaci showing strong resistance, moderate resistance or susceptibility to imidacloprid. We found that constitutive over-expression (up to approximately 17-fold) of a single P450 gene, CYP6CM1, was tightly related to imidacloprid resistance in both the B and Q biotypes. Next, we identified three single-nucleotide polymorphic (SNP) markers in the intron region of CYP6CM1 that discriminate between the resistant and susceptible Q-biotype CYP6CM1 alleles (r-Q and s-Q, respectively), and used a heterogeneous strain to test for association between r-Q and resistance. While survivors of a low imidacloprid dose carried both the r-Q and s-Q alleles, approximately 95% of the survivors of a high imidacloprid dose carried only the r-Q allele. Together with previous evidence, the results reported here identify enhanced activity of P450s as the major mechanism of imidacloprid resistance in B. tabaci, and the CYP6CM1 gene as a leading target for DNA-based screening for resistance to imidacloprid and possibly other neonicotinoids in field populations.     

Poly (ADP-ribose) polymerase inhibition synergizes with the NF-κB inhibitor DHMEQ to kill hepatocellular carcinoma cells

Poly (ADP-ribose) polymerase (PARP) enzymes play a key role in the cellular machinery responsible for DNA repair. Dehydroxymethylepoxyquinomicin (DHMEQ), a new inhibitor of NF-κB, induces oxidative stress and DNA damage. The effects of DHMEQ in combination with Olaparib (PARP inhibitor) were studied on hepatocellular carcinoma (HCC) cells. The DHMEQ–Olaparib combination synergistically inhibited cell viability, cell proliferation and colony formation of Hep3B, but had additive effects on Huh7 cells. The synergistic effects of the combination correlated with increased apoptosis, caspase 3/7 activity and PARP cleavage. There was an induction of an endoplasmic reticulum (ER) stress response with significant up-regulation of CHOP and TRB3 genes and splicing of XBP1 mRNA in Hep3B cells but not in Huh7 cells. Silencing of the TRB3 mRNA in Hep3B cells reversed the reduction in viability caused by DHMEQ–Olaparib treatment, while depletion of unspliced XBP1 mRNA in DHMEQ–Olaparib-treated Huh7 cells reduced viability. ROS production was increased after DHMEQ–Olaparib treatment of Hep3B, which caused DNA damage by an accumulation of γH2AX, increased AKT phosphorylation and reduced cell viability. The combination reduced Rad51 nuclear foci in Hep3B cells (not Huh7 cells), and silencing of Rad51 enhanced sensitivity of Huh7 cells to the DHMEQ–Olaparib combination. Knockdown of AKT in Hep3B cells restored the number of Rad51 nuclear foci after DHMEQ–Olaparib treatment. In summary, the DHMEQ–Olaparib combination induced ROS production, which killed HCC cells via DNA damage that could not be repaired by Rad51.SummaryPARPs and NF-κB are frequently deregulated in HCC. The DHMEQ–Olaparib combination exerted synergistic anti-tumour effects on HCC cells through ROS production via DNA damage that could not be repaired by Rad51. This suggested that the DHMEQ–Olaparib combination could be used to treat tumours that were resistant to Olaparib treatment.     

Control of P-glycoprotein activity by membrane cholesterol amounts and their relation to multidrug resistance in human CEM leukemia cells

P-glycoprotein (P-gp) is the most well-known ATP-binding cassette (ABC) transporter involved in unidirectional substrate translocation across the membrane lipid bilayer, thereby causing the typical multidrug resistance (MDR) phenotype expressed in many cancers. We observed that in human CEM acute lymphoblastic leukemia cells expressing various degrees of chemoresistance and where P-gp was the sole MDR-related ABC transporter detected, the amount of esterified cholesterol increased linearly with the level of resistance to vinblastine while the amounts of total and free cholesterol increased in a nonlinear way. Membrane cholesterol controlled the ATPase activity of P-gp in a linear manner, whereas the P-gp-induced daunomycin efflux decreased nonlinearly with the depletion of membrane cholesterol. All these elements suggest that cholesterol controls both the ATPase and the drug efflux activities of P-gp. In addition, in CEM cell lines that expressed increasing levels of elevated chemoresistance, the amount of P-gp increases to a plateau value of 40% of the total membrane proteins and remained unvaried while the amount of membrane cholesterol increased with the elevation of the MDR level, strongly suggesting that cholesterol may be directly involved in the typical MDR phenotype. Finally, we showed that the decreased daunomycin efflux by P-gp due to the partial depletion of membrane cholesterol was responsible for the efficient chemosensitization of resistant CEM cells, which could be totally reversed after cholesterol repletion.     

CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d''Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.     

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.     

Clitocine Reversal of P-Glycoprotein Associated Multi-Drug Resistance through Down-Regulation of Transcription Factor NF-κB in R-HepG2 Cell Line

Multidrug resistance(MDR)is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5'-serial truncation analysis of the MDR1 promoter defined a region from position -450 to -193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.     

Complex formation in vesicle-reconstituted mitochondrial cytochrome P450 systems (CYP11A1 and CYP11B1) as evidenced by rotational diffusion experiments using EPR and ST-EPR.

Rotational diffusion measurements using EPR and saturation transfer EPR were applied to analyze complex formation between the electron-transfer components of the mitochondrial steroid-hydroxylating cytochrome P450 systems (CYP11A1 and CYP11B1) in phosphatidylcholine/phosphatidylethanolamine/cardiolipin vesicles prepared by octyl glucoside dialysis/adsorption. Octyl glucoside reconstitution of P450SCC results in large vesicles, which have an advantage over small vesicles in that vesicle tumbling does not contribute to measured rotational diffusion rates. Immobilization of spin-labeled adrenodoxin by both P450SCC and adrenodoxin reductase indicates equimolar complexation between P450SCC and adrenodoxin as well as between adrenodoxin reductase and adrenodoxin. Combination of rotational diffusion and antibody cross-linking confirmed the complexation of adrenodoxin with P450SCC and for the first time provided direct evidence of a complex between P450SCC and P45011beta in the membrane. In contrast, no evidence was found for the existence of adrenodoxin reductase-P450SCC complexes or a ternary complex of all three proteins. Thus, these experiments confirm the shuttle mechanism of electron transfer to vesicle-reconstituted P450SCC and P45011beta.     

Metabolic imidacloprid resistance in the brown planthopper,Nilaparvata lugens,relies on multiple P450 enzymes

Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance.     

Histone deacetylase inhibitor apicidin-mediated drug resistance: involvement of P-glycoprotein

Multidrug resistance (MDR), which is a significant impediment to the success of cancer chemotherapy, is attributable to the overexpression of membrane transport proteins, such as P-glycoprotein (P-gp), resulting in an increased drug efflux. In this study, we show that the histone deacetylase (HDAC) inhibitor apicidin leads to resistance of HeLa cells to paclitaxel through the induction of P-gp expression. Furthermore, apicidin dramatically increases the release of a fluorescent P-gp substrate, rhodamine 123, from cells. In parallel, apicidin resistance to the apoptotic potential of paclitaxel is associated with induction of P-gp expression in HeLa cells, as evidenced by specific inhibition of P-gp function using either the pharmacological inhibitor verapamil or RNA silencing. We also demonstrate the contribution of apicidin-induced functional P-gp expression to drug resistance using KB cells. Failure of P-gp induction by apicidin does not reverse paclitaxel-induced cytotoxicity in the cells. Although HDAC inhibitors are widely appreciated as a new class of anti-tumor agent, our findings clearly demonstrate that apicidin treatment may lead to P-gp-mediated resistance to other anti-tumor agents, suggesting a need for careful design of clinical applications using HDAC inhibitors.