JNK1,JNK2,and JNK3 are involved in P-glycoprotein-mediated multidrug resistance of hepatocellular carcinoma cells

BACKGROUND:Multidrug resistance(MDR)is extremely common in hepatocellular carcinoma(HCC)and is a major problem in cancer eradication by limiting the efficacy of chemotherapy.Modulation of c-Jun NH2-terminal kinase(JNK)activation could be a new method to reverse MDR.However,the relationship between JNK activity and MDR in HCC cells is unknown.This study aimed to explore the relationship between MDR and JNK in HCC cell lines with different degrees of MDR.METHODS:A MDR human HCC cell line,SMMC-7721/ ADM,was developed by exposing parental cells to gradually increasing concentrations of adriamycin.The MTT assay was used to determine drug sensitivity.Flow cytometry was used to analyze the cell cycle distribution and to measure the expression levels of P-glycoprotein(P-gp)and MDR-related protein(MRP)-1 in these cells.JNK1,JNK2 and JNK3 mRNA expression levels were quantified by real-time PCR.Expression and phosphorylation of JNK1,JNK2,and JNK3 were analyzed by Western blotting.RESULTS:The MDR of SMMC-7721/ADM cells resistant to 0.05 mg/L adriamycin was mainly attributed to the overexpression of P-gp but not MRP1.In addition,these cells had a significant increase in percentage in the S phase,accompanied by a decrease in percentage in the G0/G1 phase,which is likely associated with a reduced ability for cell proliferation and MDR generation.We found that JNK1,JNK2,and JNK3 activities were negatively correlated with the degree of MDR in HCC cells.CONCLUSION:This study suggests that JNK1,JNK2,and JNK3 activities are negatively correlated with the degree of MDR in HCC cells.

Reversal of P-glycoprotein-mediated Multidrug Resistance in SGC7901/VCR Cells by PPARγ Activation by Troglitazone

Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR) in cancer cells.This study examined the effects of troglitazone,a ligand of peroxisome proliferator-activated receptor gamma(PPARγ),on P-gp-mediated MDR in SGC7901/VCR cells(a vincristine-resistant human gastric cancer cell line).The expression of P-gp was detected by RT-PCR and Western blotting,respectively.The SGC7901/VCR cells were treated with 0.1 mg/L vincristine(VCR) alone or in combination with 1,5,10 μmol/L troglitazone for 24 h.PPARγ was measured by electrophoretic mobility shift assay(EMSA).The intracellular concentration of Rhodamine123(Rh123,a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp.The cell cycle and apoptosis were measured by flow cytometry.The results showed that the P-gp was increasingly expressed in SGC7901,BGC823 and SGC7901/VCR cells in turn,suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp.In the SGC7901/VCR cells,the expression level of total PPARγ was increased,however,the protein level and activity of PPARγ in the nuclei of cells decreased significantly.Troglitazone elevated the PPARγ activity in SGC7901/VCR cells in a dose-dependent manner.Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner.We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G2/M phase and decreased the cell percentage in G1 and S phase in a dose-dependent manner.Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner.It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARγ activity.Elevation of the PPARγ activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells.It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.

Multidrug Resistance P-glycoprotein Function of Bone Marrow Hematopoietic Cells and the ReversalAgent Effect

The multidrug resistance P-glycoprotein (P-gp) expression and func-tion in hematopoietic stem/progenitor cells were studied to investigate whether the inhibition of hematopoietic cell P-gp function by multidrug resistance reversal agent increases the cytotoxicity of chemotherapy drugs on the hematopoietic cells.The expression of P-gp on the surface of CD cells from healthy human marrow was examined by flow cytometry. The multidrug resistance reversal agent MS-209 was used to measure the effects of MS-209 on the Rhodamin-123 uptaking o fCD hematopoietic cells. By using methylcellulose semi-solid culture, normal human granulocyte-macrophage clonal formation unit (CFU-GM) was cultured. The changes in CFU-GM inhibitory rate caused by daunorubicin were determined in the presence or absence of MS-2O9. The results showed that the P-gp expression rate of bone marrow CDL cells was 13. 3 %. MS-209 obviously increased the Rhodamin-123 uptake of CD positive cells. The mean inhibitory rate of daunorubicin for CFU-GM was 29. 6 %, but it was increased to 43. 3 % in the presence of MS-209 with the difference being significant (P< 0. 05). It was concluded that hematopoietic cells expressed P-gp protein and possessed active function- MS-209could inhibit the membrane efflux pump and increase the cytotoxicity of chemotherapy drugs to the clonal growth of hematopoeitic stem cells, suggesting the side effects of these drugs on the hematopoietic system should be taken into consideration in the clinical use.

REGULATION OF EXPRESSION OF P-GLYCOPROTEIN AND GST BY MULTIDRUG-RESISTANT REVERSORS IN ADM-SENSITIVE AND ADM-RESISTANT HUMAN TUMOR CELL LINES

Objective: To investigate the regulation of p-glycoprotein (PgP) and GST expression from three reversors in ADM-sensitive and ADM-resistant human leukemic cell lines and KB cell lines. Methods: Immunocytochemical(ICC) technique was applied to detect the multidrug-resistant gene products, PgP and GST in K562 cells, K562/ADM cells and KB cells before or after treatment with three resistant reversors, i.e., verapamil(VER), dipyriamole(DPM) and cyclosporin A(CsA). Results: PgP expression was observed in K562/ADM cells but not in K562 cells or KB cells, and GSTPI expression, in KB cells but not in K562 cells or K562/ADM cells. Overexpressions of PgP were induced after treatment with VER, or DPM or CsA for 24 h in K562 cells but not KB cells. DPM-treated K562/ADM cells expressed PgP much lower than DPM-ree of K562/ADM cells with CsA for 24 h. Induced GSTPI expression was found after treatment with DPM, but not VER or CsA in K562 cells. No significant difference was observed for GSTPI expresion in KB cells before and after treatment with VER, or DPM, or CsA. Conclusion: The findings suggested that reversal activity of some drug resistant reversors, such as VER, DPM, CsA, may be declined by themselves through induction of PgP, perhaps GST.

The multidrug resistance transporter P-glycoprotein confers resistance to ferroptosis inducers

Aim:Ferroptosis is a non-apoptotic form of cell death caused by lethal lipid peroxidation.Several small molecule ferroptosis inducers(FINs)have been reported,yet little information is available regarding their interaction with the ATP-binding cassette(ABC)transporters P-glycoprotein(P-gp,ABCB1)and ABCG2.We thus sought to characterize the interactions of FINs with P-gp and ABCG2,which may provide information regarding oral bioavailability and brain penetration and predict drug-drug interactions.Methods:Cytotoxicity assays with ferroptosis-sensitive A673 cells transfected to express P-gp or ABCG2 were used to determine the ability of the transporters to confer resistance to FINs;confirmatory studies were performed in OVCAR8 and NCI/ADR-RES cells.The ability of FINs to inhibit P-gp or ABCG2 was determined using the fluorescent substrates rhodamine 123 or purpuin-18,respectively.Results:P-gp overexpression conferred resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin.P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1.The FINs ML-162,GPX inhibitor 26a,and PACMA31 at 10µM were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells.GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells.Conclusion:Expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain.The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions.

Disease control by regulation of P-glycoprotein on lymphocytes in patients with rheumatoid arthritis

The main purpose of treatment of rheumatoid arthritis(RA) with disease modifying antirheumatic drugs(DMARDs) is to control activation of lymphocytes,although some patients do not respond adequately to such treatment. Among various mechanisms of multidrug resistance, P-glycoprotein(P-gp), a member of ATP-binding cassette transporters, causes drugresistance by efflux of intracellular drugs. Certain stimuli,such as tumor necrosis factor-α, activate lymphocytes and induce P-gp expression on lymphocytes, as evident in active RA. Studies from our laboratories showed spontaneous nuclear accumulation of human Y-boxbinding protein-1, a multidrug resistance 1 transcription factor, in unstimulated lymphocytes, and surface overexpression of P-gp on peripheral lymphocytes of RA patients with high disease activity. The significant correlation between P-gp expression level and RA disease activity is associated with active efflux of drugs from the lymphocyte cytoplasm and in drugresistance.However, the use of biological agents that reduce P-gp expression as well as P-gp antagonists(e.g., cyclosporine) can successfully reduce the efflux of corticosteroids from lymphocytes in vitro, suggesting that both types of drugs can be used to overcome drug-resistance and improve clinical outcome. We conclude that lymphocytes activated by various stimuli in RA patients with highly active disease acquire P-gpmediated multidrug resistance against corticosteroids and probably some DMARDs, which are substrates of P-gp. Inhibition/reduction of P-gp could overcome such drug resistance. Expression of P-gp on lymphocytes is a promising marker of drug resistance and a suitable therapeutic target to prevent drug resistance in patients with active RA.

Inhibition of P-glycoprotein by two artemisinin derivatives

P-Glycoprotein/MDR1 represents an important component of the blood brain barrier and contributes to multidrug resistance.We investigated two derivatives of the anti-malarial artemisinin,SM616 and GHP-AJM-3/23,concerning their ability to interact with P-glycoprotein.The ability of the two compounds to inhibit P-glycoprotein(P-gp)activity was examined in sensitive CCRF-CEM and P-gp over-expressing and multidrug-resistant CEM/ADR5000 cells as well as in porcine brain capillary endothelial cells(PBCEC)by means of calcein-AM assays.Verapamil as well-known P-gp inhibitor was used as control drug.CEM/ADR5000 cells exhibited cross-resistance to GHP-AJM-3/23,but slight collateral sensitivity to SM616.Furthermore,SM616 inhibited calcein efflux both in CEM/ADR5000 and PBCEC,whereas GHP-AJM-3/23 did only increase calcein fluorescence in PBCEC,but not CEM/ADR5000.This may be explained by the fact that CEM/ADR5000 only express P-gp but not other ATP-binding cassette transporters,whereas PBCEC are known to express several ABC transporters and calcein is transported by more than one ABC transporter.Hence,SM616 may be the more specific P-gp inhibitor.In conclusion,the collateral sensitivity of SM616 as well as the inhibition of calcein efflux in both CEM/ADR5000 cells and PBCEC indicate that this compound may be a promising P-gp inhibitor to treat cancer therapy and to overcome the blood brain barrier.

Some substrates of P-glycoprotein targeting <i>β</i>-amyloid clearance by quantitative structure-activity relationship (QSAR)/membrane-interaction (MI)-QSAR analysis

The pathogenesis of Alzheimer’s disease (AD) putatively involves a compromised blood-brain barrier (BBB). In particular, the importance of brain-to-blood transport of brain-derived metabolites across the BBB has gained increasing attention as a potential mechanism in the pathogenesis of neurodegenerative disorders such as AD, which is characterized by the aberrant polymerization and accumulation of specific misfolded proteins, particularly β-amyloid (Aβ), a neuropathological hallmark of AD. P-glycoprotein (P-gp), a major component of the BBB, plays a role in the etiology of AD through Aβ clearance from the brain. Our QSAR models on a series of purine-type and propafenone-type substrates of P-gp showed that the interaction between P-gp and its modulators depended on Molar Refractivity, LogP, and Shape Attribute of drugs it transports. Meanwhile, another model on BBB partitioning of some compounds revealed that BBB partitioning relied upon the polar surface area, LogP, Balaban Index, the strength of a molecule combined with the membrane-water complex, and the changeability of the structure of a solute-membrane-water complex. The predictive model on BBB partitioning contributes to the discovery of some molecules through BBB as potential AD therapeutic drugs. Moreover, the interaction model of P-gp and modulators for treatment of multidrug resistance (MDR) indicates the discovery of some molecules to increase Aβ clearance from the brain and reduce Aβ brain accumulation by regulating BBB P-gp in the early stages of AD. The mechanism provides a new insight into the therapeutic strategy for AD.

Evaluating the regulation of transporter proteins and P-glycoprotein in rats with cholestasis and its implication for digoxin clearance

BACKGROUND Cardiac and hepatic functionality are intertwined in a multifaceted relationship.Pathologic processes involving one may affect the other through a variety of mechanisms,including hemodynamic and membrane transport effects.AIM To better understand the effect of extrahepatic cholestasis on regulations of membrane transporters involving digoxin and its implication for digoxin clearance.METHODS Twelve adult rats were included in this study;baseline hepatic and renal laboratory values and digoxin pharmacokinetic(PK)studies were established before evenly dividing them into two groups to undergo bile duct ligation(BDL)or a sham procedure.After 7 d repeat digoxin PK studies were completed and tissue samples were taken to determine the expressions of cell membrane transport proteins by quantitative western blot and real-time polymerase chain reaction.Data were analyzed using SigmaStat 3.5.Means between pre-surgery and post-surgery in the same experimental group were compared by paired t-test,while independent t-test was employed to compare the means between sham and BDL groups.RESULTS Digoxin clearance was decreased and liver function,but not renal function,was impaired in BDL rats.BDL resulted in significant up-regulation of multidrug resistance 1 expression in the liver and kidney and its down-regulation in the small intestine.Organic anion transporting polypeptides(OATP)1A4 was up-regulated in the liver but down-regulated in intestine after BDL.OATP4C1 expression was markedly increased in the kidney following BDL.CONCLUSION The results suggest that cell membrane transporters of digoxin are regulated during extrahepatic cholestasis.These regulations are favorable for increasing digoxin excretion in the kidney and decreasing its absorption from the intestine to compensate for reduced digoxin clearance due to cholestasis.

Antiepileptic drug-induced multidrug resistance P-glycoprotein overexpression in astrocytes cultured from rat brains

Background Intractable epilepsy may be due to multidrug resistance induced by conventional antiepileptic drugs. The phenomenon is sometimes associated with an overexpression of multidrug resistance gene 1 (MDR 1). The purpose of this study was to determine if the overexpression of MDR 1 could be induced in astrocytes from rat brains in vitro using antiepileptic drugs.Methods Astrocyte cell cultures from postnatal Wistar rats (within 24 hours of birth) were established. Different concentrations of the antiepileptic drugs phenytoin, phenobarbital, carbamazepine, and valproic acid were added to the cultures for 10, 20, or 30 days. The expression of P-glycoprotein (Pgp), the protein product of MDR 1, was investigated with immunocytochemistry. Results Less than 5% of normal, untreated astrocytes had detectable Pgp staining at any time point. Phenytoin, phenobarbital, carbamazepine, and valproic acid induced the overexpression of Pgp in astrocytes in a dose- and time-dependent manner. Significantly higher levels of Pgp staining were detected at therapeutic concentrations of certain antiepileptic drugs (20 μg/ml phenobarbital, 40 μg/ml phenobarbital, and 20 μg/ml phenytoin) on day 30. Upregulation of Pgp was detected when using higher concentrations of phenytoin, phenobarbital, and valproic acid on day 20 and when using higher concentrations of any of the four antiepileptic drugs on day 30. Conclusions Treatment with antiepileptic drugs may contribute to the overexpression in astrocytes of MDR 1 and its protein product, Pgp. The mechanism leading to MDR must be considered in patients undergoing long-term treatment with antiepileptic drugs.

Effects of Jianpi Jiedu Recipe (健脾解毒方)on Reversion of P-Glycoprotein-Mediated Multidrug Resistance through COX-2 Pathway in Colorectal Cancer

Objective： To evaluate the underlying mechanism of Jianpi Jiedu Recipe （健脾解毒方, JJR） in the reversion of multidrug resistance concerning colorectal cancer in vitro and in vivo. Methods： Mice were treated orally with JJR at a daily 4.25 g/（kg.day） or injected with vinblastine （VCR） 2.5 mg/（kg day） for 3 weeks after having been inoculated with HCT8N cells; tumor tissues were assayed by hematoxylin and eosin staining. Firstly, the effects of JJR on the expression of cyclooxygenase-2 （COX-2） were tested by real-time polymerase chain reaction （PCR） technique and COX-2 gene silenced by siRNA. Secondly, the variation of intracellular concentration of oxaliplatin （L-OHP） was evaluated by the inductively coupled plasma mass spectroscopy （ICP- MS） in HCT8N and its COX-2 siRNA cells; the concentration of J JR combined with chemotherapeutic drugs and the reverse effect of multidrug resistance （MDR） in HCT8N cells was evaluated by the MTT assay. Thirdly, real-time quantitative PCR and Western blot analysis were used to detect the multidrug resistance gene 1 （MDR1） mRNA and P-gp expression. Results： JJR had an inhibitory effect on the growth of tumors in vivo, and it, in combination with chemotherapeutic drugs, could reverse the drug-resistance of HCT8N cells and increase the sensitivity of HCT8N cells to VCR, DDP, 5-Fu, and THP. ICP-MS results showed that JJR could increase the concentration of drugs in HCT8/V cells （P〈0.01）. Furthermore, it was shown that JJR could reverse drug resistance of colorectal cancer cells by decreasing MDR1 expression and P-gp level via downregulation of COX-2, which has been represented as one of the major mechanisms that contributes to the MDR phenotype （P〈0.01）. Conclusion： JJR reversed multidrug resistance and enhanced the sensitivity to chemotherapy, which could be attributed to the down-regulation of COX-2 in MDRl/P-gp-mediated MDR colorectal cancer after chemotherapy.

Reversion effects of curcumin on multidrug resistance of MNNG/HOS human osteosarcoma cells in vitro and in vivo through regulation of P-glycoprotein

Background P-glycoprotein （P-gp） encoded by ATP-binding cassette sub-family B member 1 （ABCB1） gene is a kind of ATP-dependent drug transporter, which plays important roles in multidrug resistance （MDR） of human cancers, such as osteosarcoma. Curcumin is a natural phenolic coloring compound originating from the rhizomes of Curcuma Ionga, which is proved to possess antitumor biological activities including reversion of MDR. However, the effect and molecular mechanisms of curcumin to osteosarcoma MDR remain unclear.

The roles of the human ATP-binding cassette transporters P-glycoprotein and ABCG2 in multidrug resistance in cancer and at endogenous sites: future opportunities for structure-based drug design of inhibitors

The ATP-binding cassette(ABC)transporters P-glycoprotein(P-gp)and ABCG2 are multidrug transporters that confer drug resistance to numerous anti-cancer therapeutics in cell culture.These findings initially created great excitement in the medical oncology community,as inhibitors of these transporters held the promise of overcoming clinical multidrug resistance in cancer patients.However,clinical trials of P-gp and ABCG2 inhibitors in combination with cancer chemotherapeutics have not been successful due,in part,to flawed clinical trial designs resulting from an incomplete molecular understanding of the multifactorial basis of multidrug resistance(MDR)in the cancers examined.The field was also stymied by the lack of high-resolution structural information for P-gp and ABCG2 for use in the rational structure-based drug design of inhibitors.Recent advances in structural biology have led to numerous structures of both ABCG2 and P-gp that elucidated more clearly the mechanism of transport and the polyspecific nature of their substrate and inhibitor binding sites.These data should prove useful helpful for developing even more potent and specific inhibitors of both transporters.As such,although possible pharmacokinetic interactions would need to be evaluated,these inhibitors may show greater effectiveness in overcoming ABC-dependent multidrug resistance in combination with chemotherapeutics in carefully selected subsets of cancers.Another perhaps even more compelling use of these inhibitors may be in reversibly inhibiting endogenously expressed P-gp and ABCG2,which serve a protective role at various blood-tissue barriers.Inhibition of these transporters at sanctuary sites such as the brain and gut could lead to increased penetration by chemotherapeutics used to treat brain cancers or other brain disorders and increased oral bioavailability of these agents,respectively.

Carbonic anhydrase XII inhibition overcomes P-glycoprotein-mediated drug resistance: a potential new combination therapy in cancer

Intrinsic or acquired resistance to chemotherapy is a major hurdle in the treatment of cancer.One of the key mechanisms of resistance is the overexpression of the drug efflux transporter P-glycoprotein(Pgp).Pgp overexpression renders a large number of mechanistically unrelated chemotherapies ineffective.Targeting Pgp inhibition directly to overcome drug resistance,although conceptually and mechanistically attractive,has not translated to the clinic,in part because Pgp also has a critical protective function in many healthy tissues.It was recently discovered that carbonic anhydrase XII(CA XII),an enzyme associated with pH regulation in cancer,is co-expressed and co-located with Pgp in drug resistant cancer cells.CA XII is also upregulated by hypoxia,which is another microenvironmental factor that contributes to drug resistance.Here,we review findings that demonstrate modulation of CA XII may offer a promising new approach towards overcoming the longstanding hurdle of drug resistance and therapy failure against solid cancers.This review covers the use of CA XII inhibitors,both small molecule and antibody,in combination with chemotherapeutics that are substrates for Pgp.This combination therapy approach restores the efficacy of chemotherapy in resistant cells and offers a potential new therapeutic window to re-examine the targeting of Pgp as a safe,effective,and novel anticancer strategy.

Effects of arsenic trioxide on drug transporting molecules in multidrug resistance malignant neoplasma MR2 cell line

Objective： To study the effect of arsenic trioxide （As203） on the expression of drug transporting molecules in multidrug resistance malignant neoplasma acute promyelocytic leukemia （APL） MR2 cell line. Methods： MR2 resistant to alltrans retinoic acid （ATRA） and non-ATRA resistant APL cell line NB4 were used. Expressions of P-glycoprotein （Pgp）, multidrug resistance protein （MRP） and lung resistance-related protein （LRP） were detected by immunocytochemical assay. Results： The expression of Pgp was significantly higher in MR2（30%-40%） than that in NB4（10%-20%） （P 〈 0.001）, and the expression of MRP was also higher in MR2 （56.9 ± 3.4 - 21.2 ± 1.1） than that in NB4 （20.6 ± 5.3 - 16.7 ± 1.2） （P 〈 0.001）. As2O3 ranging from 0.5-2.0 μmol/L, could significantly decrease the expressions of Pgp and MRP. The expression of Pgp and MRP in MR.2 cell line were negatively correlated with the dose and duration of action of As2O3. Conclusion： Pgp and MRP may be the sensitive targets of As2O3 to overcome drug-resistance. ATRA might be the substrates of Pgp and MRP.

Arsenic trioxide inhibits p-glycoprotein expression in multidrug-resistant human leukemia cells that overexpress the MDR1 gene

Objective To investigate the effects of arsenic trioxide (As2O3) on the apoptosis and p-glycoprotein (P-gp) expression of multidrug-resistant human leukemia cells.Methods Human multidrug-resistant leukemia cell line K562/ADM overexpressing the MDR1 gene, was used as the target cells. The cell proliferating activity was assessed using the MTT colorimetric assay. Cytomorphology was investigated under light, confocal and electron microscopes. DNA fragmentation was examined using agarose gel electrophoresis, while p-gp expression, cell cycle status and sub-G1 cells were determined using flow cytometry.Results Zero point five to 20 μmol/L As2O3 inhibited the proliferation of K562/ADM cells, and K562/ ADM cells were more sensitive to As2O3 than the parental K562 cells. As2O3-induced apoptosis of K562/ADM cells was determined by the observance of typical morphological changes and the appearance of DNA ladder and sub-G1 cell populations. As2O3 significantly inhibited the P-gp expression of K562/ADM cells, and synergistically enhanced the sensitivity of the drug-resistant cells to adriamycin.Conclusions As2O3 induces growth-inhibition and apoptosis, down-regulates P-gp expression and exerts a synergistic effect in combination with adriamycin in multidrug-resistant leukemia cells.

Synergistic Effect of Hyperthermia and Neferine on Reverse Multidrug Resistance in Adriamycin-resistant SGC7901/ADM Gastric Cancer Cells

Multidrug resistance（MDR） plays a major obstacle to successful gastric cancer chemotherapy.The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine（Nef） in adriamycin（ADM） resistant human SGC7901/ADM gastric cancer cells.The MDR cells were heated at 42℃ and 45℃ for 30 min alone or combined with 10 μg/mL Nef.The cytotoxic effect of ADM was evaluated by MTT assay.Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique.Intracellular accumulation of ADM was monitored with high performance liquid chromatography.Mdr-1 mRNA,P-glycoprotein（P-gp）,γH2AX expression and γH2AX foci formation were determined by real-time PCR,Western blot and immunocytochemical staining respectively.It was found that different heating methods induced different cytotoxic effects.Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells.The water submerged hyperthermia increased the cell membrane fluidity.Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM.The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp.The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells.The higher temperature was,the worse effect was.Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.

Effects of multidrug resistance, antisense RNA on the chemosensitivity of hepatocellular carcinoma cells

BACKGROUND: Multidrug resistance is a major obstacle in cancer chemotherapy. We examined whether the antisense RNA of multidrug resistance gene 1 (mdr1) could reverse multidrug resistance in the human hepatocellular carcinoma (HCC) cell line SMMC7721/ADM. METHODS: The recombinant adenoviruses pAdEasy- GFP-ASmdr1 product was produced by the adenoviral vector AdEasy system, which can express antisense RNA against the mdr1 gene. Following that, the recombinant adenovirus was transfected into the P-glycoprotein- producing multidrug resistance cell line, SMMC7721/ADM human HCC cells resistant to adriamycin (ADM) and daunorubicin (DNR). In order to investigate the reversal of multidrug resistance phenotype, we measured the expression of mdr1 mRNA by RT-PCR and the production of P-glycoprotein by flow cytometry. The sensitivities for ADM and DNR SMMC7721/ADM cells were examined by [3-(4, 5-dimethylthi-azol-2-yl)-2,5 diphenyl-terazolium bromide] (MTT) analysis. RESULTS: The low-level expression of mdr1 mRNA and P-glycoprotein production were observed in parental sensitive cells SMMC/7721 in addition to the overexpressionof mdr1 mRNA and P-glycoprotein in SMMC7721/ADM cells. The transfection of antisense-RNA into SMMC7721/ ADM cells resulted in decreases of mdr1 mRNA and P-glycoprotein, but increase of drug sensitivities. The sensitivities of transfected SMMC7721/ADM cells to ADM and DNR in IC50 reduced by 31.25% and 62.96% respectively. CONCLUSIONS: Mdr1 antisense RNA can increase the sensitivities of SMMC7721/ADM cells to anticancer drug by decreasing the expression of the mdr1 gene and inhibiting P-glycoprotein expression. This strategy may be applicable to cancer patients with P-glycoportein mediated multidrug resistance.

β-escin reverses multidrug resistance through inhibition of the GSK3β/β-catenin pathway in cholangiocarcinoma

AIM: To develop a safe and effective agent for cholangiocarcinoma(CCA) chemotherapy. METHODS: A drug combination experiment was conducted to determine the effects of β-escin in c o m b i n a t i o n w i t h c h e m o t h e ra p y o n C C A c e l l s. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed to determine the effects of β-escin and common chemotherapeutics on the proliferation of human CCA cells(QBC939, Sk-Ch A-1, and MZ-Ch A-1). Immunocytochemistry was used to detect the expression of P-glycoprotein(P-gp) protein. Luciferase reporter assay was used to detect the activation of the Wnt/β-catenin pathway. The protein levels of P-gp, p S9-GSK3β, p T216-GSK3β, GSK3β, β-catenin, and p-β-catenin were further confirmed by western blotting.RESULTS: The drug sensitivity of QBC939 and QBC939/5-fluorouracil(5-FU) cells to 5-FU, vincristine sulfate(VCR), or mitomycin C was significantly enhanced by β-escin compared with either agent alone(P < 0.05). In addition, the combination of β-escin(20 μmol/L) with 5-FU and VCR was synergic with a combination index < 1. Further investigation found that the m RNA and protein expression of P-gp was downregulated by β-escin. Moreover, β-escin induced GSK3β phosphorylation at Tyr-216 and dephosphorylation at Ser-9, resulting in phosphorylation and degradation of β-catenin. Interestingly, activation of the GSK3β/β-catenin pathway induced by Wnt3 a resulted in upregulation of P-gp, which was effectively abolished by β-escin, indicating that β-escin down-regulated P-gp expression in a GSK3β-dependent manner.CONCLUSION: β-escin was a potent reverser of P-gpdependent multidrug resistance, with said effect likely being achieved via inhibition of the GSK3β/β-catenin pathway and thus suggesting a promising strategy of developing combination drugs for CCA.

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

AIM： 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. METHODS： 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.RESULTS： 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）. CONCLUSION： RB intrinsically expresses GSTπ, P-gp and LRP. GSTπ expression is positive in 100% welldifferentiation 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.