Objectives: Multi-drug resistance (MDR) to chemotherapy remains a major obstacle to overcome in the successful treatment of patients with cancers. It was recently discovered that Leptomycin B (LMB) reduces the paclitaxel-induced MDR in CCRF-CEM/Taxol cells. However, the mechanism remains unclear. Here we sought to explore the mechanism of LMB to reduce the MDR induced by paclitaxel. Results: LMB has remarkable cytotoxic effects in both sensitive CCRF-CEM and resistant CCRF-CEM/Taxol cell lines. The paclitaxel-induced MDR was reduced by 0.013 μm of LMB. Lower concentration of LMB regulated cell cycle progress, in situ expressions of P-gp, MRP1, and LRP, expression of CRM1, and localization of P-gp and CRM1 in CCRF-CEM/taxol cells. Study Design: Cytotoxicity of LMB on cancerous cell lines was determined by MTT assay. Cell cycle progress and in situ expressions of P-gp, MRP1, and LRP were analyzed by flow cytometry. Expression of CRM1 in the cells was examined by Western blot. And co-localization between P-gp and CRM1 was determined by laser confocal microscopy. Conclusion: The paclitaxel-induced MDR of CCRFCEM/Taxol cells was reduced by lower concentration of LMB. The mechanisms might be related to decreasing in situ expression of drug transporter proteins, promoting cell cycle progress, and altering co-localization between P-gp and CRM1 in the resistant cells.
Acquisition of multi-drug resistance (MDR) is the leading cause of treatment failure in cancer therapy. MDR is, in large part, a story of drug transporters [
Acquired drug resistance can manifest in many ways: for example, cell signaling pathways can arrest cell cycle and delay apoptosis [
Leptomycin B (LMB) is an unsaturated; branchedchain fatty acid originated from Streptomyces sp., and was firstly discovered as an antifungal agent. Recently, some studies addressed that it can serve as a specific inhibitor of the substrates containing Nuclear Export Signal sequence (NESs) and block the molecules to export from nucleus, such as HIV-1 Rev protein and Revdependent mRNA [10,11], transcription factor E2F4 [
Purpose of the present study was to explore the reversal effect of LMB on MDR induced by paclitaxel. By means of investigating the cytotoxicity of LMB on cancerous cell lines and examining the cellular events cell cycle progress, in situ expression of drug transporter protein P-gp, MRP1, and LRP, expression of CRM1, and colocalization between P-gp and CRM1 to explore the mechanisms of LMB for the effects. Results from the current study demonstrated that LMB reduces the cancerous MDR induced by paclitaxel and enhances the sensitivity of CCRF-CEM/Taxol cells to chemotherapeutic drugs.
Mouse primary monoclonal antibodies to P-gp, MRP1, and LRP were purchased from Sigma-Aldrich (St. Louis, USA). Rabbit primary polyclonal antibody to CRM1 and rabbit polyclonal antibody to β-actin (HRP) were from Abcam (Cambridge, UK). Mouse monoclonal antibody iso-type matched reagents and goat anti-mouse IgG (Fc specific) antibody conjugated with FITC were from Sigma-Aldrich. Goat anti-mouse IgG (H + L) Alexa Fluor® 488 and goat anti-rabbit IgG (H + L) Alexa Fluor® 555 were from Molecular probes (Eugene, USA). Horse-radish peroxidase (HRP) conjugated goat anti-rabbit IgG (H + L) was from SouthernBiotech (Birmingham, USA).
Multi-drug resistant CCRF-CEM/Taxol cells induced by paclitaxel and its parental sensitive CCRF-CEM (Acute Lymphoblastic Leukemia, T-cell, Human) cells were maintained in RPMI-1640 media supplemented with 10% (v/v) fetal bovine serum (FBS, from Biochrom, Berlin). The cancer cells were cultured in a humidified 5% CO2 /95% air incubator at 37˚C [
Exponentially growing CCRF-CEM/Taxel and CCRFCEM cells were harvested and seeded in 24-well plates at a density of 3 to 6 × 104 cells/500μl/well in complete medium. The first column of wells was exposed to DMSO as negative control. The other 5 columns of wells were exposed to different concentration of LMB or chemotherapeutic drugs as testing groups. The antiproliferative effect of the drugs was evaluated after 48 hrs by MTT Assay. All the studies were conducted in triplicate for each sample concentration.
Drug resistance of CCRF-CEM/Taxol cells was evaluated using the resistance fold calculated by the ratio of IC50 values of resistant cells versus that of its parental sensitive CCRF-CEM cells.
For examination of the effects of LMB on the paclitaxel-induced MDR of CCRF-CEM/Taxol cells, the cells were exposed to different concentration of chemotherapeutic drugs simultaneously with a lower concentration of LMB. The effects were evaluated using decreasing the resistance folds of CCRF-CEM/Taxol cells to different chemotherapeutic drugs.
To quantitatively assess in situ expression of the drug transporter proteins in CCRF-CEM and CCRF-CEM/Taxol cells, the cells were harvested and fixed with 2% formaldehyde in PBS at room temperature for 10 min, washed with PBS (containing saponin:BSA:sodium azide = 0.04%:0.1%:0.01%) twice, then were incubated with mouse primary monoclonal antibodies to P-gp, MRP1 and LRP, respectively. Washed with PBS (containing 0.04% saponin) twice, the cells were incubated with goat anti-mouse IgG (FITC) and washed with PBS in presence of saponin twice and in absence of saponin once prior to be fixed in 1% paraformaldehyde. To account for nonspecific background fluorescence, cells were incubated with murine IgG1 antibody as control and backgro-0 und fluorescence intensity was subtracted from specific signals [20,21]. The flow cytometry analysis was performed on a FACSCalibur with CellQuest Pro v5.2.1. (Becton Dickinson). All the studies were conducted in triplicate.
For assessment of P-gp expression on the membrane of CCRF-CEM and CCRF-CEM/Taxol cells, the cells were processed according to the method described above just in absent of saponin.
For cell cycle analysis, CCRF-CEM/Taxol cells were treated with LMB as testing groups paralleled with untreated CCRF-CEM/Taxol cells as positive control and with CCRF-CEM cells as negative control, respectively. Cell cycle analyses were carried out after the cells were synchronized and cultured for 48 h. The cell populations were fixed in 70% of ice-cold ethyl alcohol for 3 h, treated with 5 u/μl of RNase H solution at 37˚C for 30 min, stained with 50 μg/ml of propidium iodide for 15 min, and then subjected to flow cytometry with a FACScan flow cytometer (Becton Dickinson). 10,000 events were collected from each sample. Data acquisition was carried out using Cell-Quest software and cell cycle distribution calculated using the ModFit LT 3.0 (Verity Software House, ME, USA) analysis software [
To explore the expression of CRM1 affected by lower concentration of LMB in CCRF-CEM/Taxol cells, the cells were treated with 0.013 and 0.015 μm of LMB and compared to CCRF-CEM cells as negative control and CCRF-CEM/Taxol cells as positive control. The whole cell lysates were prepared from both CCRF-CEM and CCRF-CEM/Taxol cell line cancerous cells. The protein concentration of lysates was determined with 2-D Quant Kit (Amersham Biosciences), and 30 μg proteins were applied to a 10% SDS-polyacrylamide gel and electrophoresed for 90 min at 100 V. The protein was then transferred to nitrocellulose membrane (Millipore). After blocking with 5% of skimmed milk in PBST (PBS-0.2% Tween 20) overnight at 4˚C, the membranes were incubated together with rabbit primary antibodies to CRM1 for 3 hrs at 37˚C. HRP-conjugated goat anti-rabbit IgG (HRP) served as the secondary antibody. The membranes were briefly incubated with enhanced ECL detection reagent (SuperECL Plus, Applygen Technolgies Inc.) to visualize the protein and exposed to an X-ray film. β-actin served as the internal control. Density of plot on lanes was quantified using Image J software (version 1.33).
For confocal analysis to visualize the effect of LMB on intracellular localization of CRM1 and P-gp in CCRFCEM/Taxol cells, the cells and negative CCRF-CEM cells were grown in phenol red-free RPMI-1640 media for 48 hrs and fixed with 70% ethanol for 15 min at room temperature, permeated with 0.04% saponin for 5 mins, then reacted with mouse monoclonal primary antibody to P-gp and rabbit polyclonal primary antibody to CRM1 for 2 hrs at 37˚C. The cells were stained by goat antimouse IgG conjugated with Alexa Fluor 488 (green, Molecular Probes) and goat anti-rabbit IgG conjugated with Alexa Fluro 555 (red, Molecular Probes), then were mounted with DAPI (blue) to detect nucleus. Confocal microscopy was performed on a Zeiss LSM 510 META system (60 × oil-immersion objectives) equipped with a 150 Mw Omnichrome Ar-Kr laser exciting at 488 nm and 555 nm. Images were captured using Carl Zeiss Laser Scanning Microscope LSM 510 Release (Version 4.2).
Statistical analysis in this study were carried out with OriginPro 8 SR0 (v. 8.0724) software. The cell fraction of cell cycle progression and in situ expression of the drug transporter proteins were evaluated by 2-way ANOVA followed by Student’s t-test. Differences with P values of less than 0.05 were considered to be statistically significant.
To determine the cytotoxicity of LMB, CCRF-CEM/ Taxol and CCRF-CEM cells were treated with different concentration of LMB. The antiproliferative effect of LMB was observed after treating the cells with LMB for 48 hrs (
CCRF-CEM/Taxol cells are resistant to paclitaxel, vinblastin, and daunorubicin simultaneously. Treatment with 0.013 μm of LMB for 48 hrs decreased the drug resistance of CCRF-CEM/Taxel cells to paclitaxel from 250.5- to 125.1-fold (at 50.06%) (
To examine the effects of LMB on cell cycle progresssion, flow cytometric analysis was performed. Compared to CCRF-CEM cells, the amount of G0/G1 and G2/M phase cells of CCRF-CEM/Taxol are larger, and the amount of S phase and apoptosis cells are lesser (
The effect of LMB treatment to enhance the expression of CRM1 in CCRF-CEM/Taxol cells was determined at the protein level, using Western blotting on protein extracts from LMB treated CCRF-CEM/Taxol cells compared to CCRF-CEM cells as negative control. CRM1 in CCRF-CEM/Taxol cells was found to be expressed at lower level compared to that in CCRF-CEM cells, as demonstrated in
Quantitative analysis of immnofluorescence staining by flow cytometry revealed that drug transporter proteins P-gp, MRP1, and LRP were expressed in both CCRFCEM/Taxol and CCRF-CEM cells. CCRF-CEM/Taxol cells expressed high levels of the three proteins at 15.4- fold to P-gp, 3.08-fold to MRP1, and 2.19-fold to LRP compared to CCRF-CEM cells (
To further clarify the effect of LMB on P-gp, the expression and distribution of P-gp in CCRF-CEM/Taxol cells were determined following 48 and 96 h of LMB exposure. Flow cytometry analysis revealed that CCRFCEM/Taxol cells expressed significantly higher level of P-gp at total cellular amount and on membrane compared to CCRF-CEM cells (
Colocalization studies between P-gp and CRM1 for intracellular compartments were performed to investigate whether sequestration plays a role in any of the drug resistance phenotypes. The stronger colocalization was observed between P-gp and CRM1 in cytoplasm of CCRFCEM cells (
Multi-drug resistance to chemotherapy remains a major obstacle to overcome in the successful treatment of patients with cancers. Due to the inherent side effect of many therapeutic drugs, the resistance presented by cancerous cells cannot be overcome by simply increasing doses to cancer targets. Many anticancer drugs have a steep dose-toxicity curve. Therefore, changes in the doses of drugs will likely have significant impacts on the clinical responses to drug treatments.
Leptomycin B (LMB) was originated from the actinomycetes of Streptomyces. It was reported as a cytotoxic agent inducing cell apoptosis [
and LRP) and expression of nuclear export protein CRM1, cell cycle progression, colocalization between P-gp and CRM1 in the resistant cells.
Increasing drug efflux predominantly via ATP-driven extrusion pumps frequently of the ATP-banding cassette (ABC) superfamily may be the major of modalities of chemotherapeutic drug resistance. P-glycoprotein (P-gp) and multi-drug resistance protein 1 (MRP1) belong to the ABC superfamily members. Lung cancer resistance-related protein (LRP) is not the member of ABC transporter proteins. LRP corresponds to the human major cytoplasmic vault protein composed of 4 proteins and one small RNA molecule, which have been hypothesized to be involved in nucleo-cytoplasmic transport of drugs [3,24]. These pumps significantly decrease the intracellular concentration of a multitude of endogenous and exogenous cytotoxic agents, thereby resulting the cancer cells in MDR [25-27]. Higher the proteins expression was found to be strongly correlation with intrinsic resistance to chemotherapeutic drugs. In the present study, the three proteins expression level were detected in LMB-treated and untreated CCRF-CEM/Taxol cells. The established paclitaxel-induced CCRF-CEM/Taxol cells had higher level of the proteins than its parental CCRF-CEM cells, and its MDR to drugs induced by paclitaxel in the cell viability assay (
The P-gp localizing on cell membrane plays a role of drug efflux pump, which directly contributes to export of chemotherapeutic drugs from cancerous cells, which is responsible for MDR phenotype of cancerous cells, in spite of the chemotherapeutic drugs are a wide variety of structurally and functionally unrelated with cytotoxicity [
Another possible mechanism for LMB-mediated MDR is the regulation of cell cycle progression. CRM1 is the major cellular target of LMB, a nuclear protein that shuttles between nucleus and cytoplasm as a nuclear-cytoplasmic transporter for a NES-mediated nuclear export pathway [11,29,30]. On the other hand, CRM1 inactivates one or more components of the pocket protein G1 arrest pathway. CRM1-dependent nuclear export regulates the G1 arrest function of E2F4 and E2F5. Over-expression of CRM1 prevents cell cycle G1 arrest by an E2F4-dependent process [
Colocalization between CRM1 and P-gp was observed in chemotherapeutic drug sensitive CCRF-CEM cells. No significant overlap of the two proteins occurred in paclitaxel-induced drug resistant CCRF-CEM/Taxol cells. Further studies will be carrying out to explore the mechanism of colocalization between CRM1 and P-gp in MDR phenotype.
In summary, we detected LMB cytotoxicity in cancerous cell lines and the effects of LMB on in situ expression of three and expression of one drug transporter proteins, cell cycle progression, and colocalization between P-gp and CRM1 in drug sensitive CCRF-CEM cells and paclitaxel-induced drug resistant CCRF-CEM/ Taxol cells. Our data demonstrated that LMB reduces paclitaxel-induced MDR and enhances susceptibility of drug resistant CCRF-CEM/Taxol cells to chemotherapeutic drugs. The present work indicates that LMB renders CCRF-CEM/Taxol cells with decreased MDR to paclitaxel, vinblastin and daunorubicin. We are confident that the effect of LMB is most likely related to decreasing in situ expression level of drug transporter proteins P-gp, MRP1, and LRP, regulating cell cycle progression, and altering the localization between P-gp and CRM1 proteins in CCRF-CEM/Taxol cells.
The study was supported by the grant from Department of Drug Safety Evaluation and Research of Beijing Institute of Pharmacology and Toxicology. We thank Dr. Chou Ting-Chao for the generous gifts of cancerous cell lines.