Clinically, multiple-drug resistance (MDR) is one of the major causes for the failure of conventional chemotherapeutic treatment of cancer. MDR severely limits the effectiveness of chemotherapy in a variety of common malignancies and it is responsible for the poor overall efficacy of cancer chemotherapy.1,2 Several strategies for overcoming drug resistance have been studied, one of which is to develop a variety of reversal agents such as verapamil, cyclosporin A (CsA), and so on.1,3 However, the outcomes of clinical trials using these drugs are very disappointing due to their dose-limiting toxicity. Development of novel anticancer drugs insensitive to or bypassing the MDR mechanisms is currently a major focus of research.4
MDR is often associated with the expression of P-glycoprotein (P-gp), which functions as a drug efflux pump to unilaterally transport intracellular drugs out of the cells, consequently conferring drug resistance to tumor cells.5–7 Much researches have focused on the function of P-gp and on ways to overcome P-gp-mediated MDR, thereby increasing the intracellular accumulation of chemotherapeutic agents.8 Recently, the development of novel drugs insensitive to or bypassing MDR mechanisms is another strategy for the treatment of MDR tumor therapy, which may provide new insight into alternative ways to overcome MDR in cancer chemotherapy.
Natural products are sources of novel compounds for fighting MDR tumor cells. Solamargine (SM), a natural steroidal alkaloid glycoside compound that reportedly exhibits strong cytotoxicity (Figure 1), induces apoptosis, and synergistically enhances the effects of chemotherapeutic agents in several human cancer cell lines.9,10 In this report, we further demonstrated the significant cytotoxicity of SM against MDR tumor cell lines. An adriamycin- resistant K562/A02 cell line that overexpresses P-gp was chosen as the model system to investigate the ability of SM to induce apoptosis and the underlying molecular mechanisms in MDR cells.
SM, isolated from Solanum incanum, a Chinese medicinal plant, was produced by the Nature Products Department of School of Pharmaceutical Sciences, Shandong University with its purity of more than 98%. It was initially dissolved in dimethyl sulfoxide (DMSO) to obtain desired concentrations for experimental use. The reference drugs, adriamycin, cisplatin, vincristine (VCR) and paclitaxel (Taxol) were purchased from Pharmacia Italia S.p.A (Italy), Qilu Pharmaceutical Factory (China), Zhejiang Haizheng Pharmaceutical Factory (China) and Beijing Xiehe Pharmaceutical Factory (China), respectively. 3-(4,5-Dimethylthiazol)-2,5- diphenyltetrazolium bromide (MTT) and 14′-2;,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Co., USA. All other chemicals used in the experiments were commercial products of reagent grade.
The human myelogenous leukemia K562 cell line and its multidrug-resistant counterpart K562/A02 were obtained from the Department of Pharmacology, the Institute of Hematology of Chinese Academy of Medical Sciences (Tianjin, China). The human NSCLC H460 cells and its multidrug-resistant counterpart H460/Taxol were kindly gifted by Dr. YAN Bing, Shandong University. Squamous cell carcinoma KB parental cell lines were obtained from the American Type Culture Collection (Rockville, MD), The VCR-selected MDR KB/VCR subline was obtained from Zhongshan University of Medical Sciences (Guangzhou, China). K562/A02 cells were maintained in a complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin at 37°C in a humidified atmosphere of 5% CO2. The cells were cultured for two weeks in drug-free medium prior to their use in the experiments.
The cytotoxicity of SM, VCR, Taxol, adriamycin, cisplatin was evaluated in K562, KB and H460 cells and their MDR counterpart cells K562/A02, KB/VCR and H460/Taxol cells by the MTT assays. Cells were seeded into 96-well plates and treated with various concentrations of the test compounds and incubated for 24 hours. Then, MTT 5 mg/ml was added to each well for 4 hours, and the resulting crystals were dissolved in DMSO. Optical density was measured by MTT assay. Cell inhibitory ratio (%) = (A570sample-A570blank)/ (A570control-A570blank)×100%. The values of concentration resulting in 50% inhibition of cell growth (IC50) were calculated from plotted results using untreated cells as 100%. The resistance factor was calculated as the ratio of the IC50 value of the MDR cells to that of corresponding sensitive parental cells.
Staining with DAPI
Cells were seeded on 12 mm round glass coverslips (precoated with poly-l-lysine) and placed at the bottom of the 24-well plates. Twelve hours after SM treatment, cells were washed once in phosphate buffer saline (PBS) followed by fixation with cold methonal:acetone (1:1) for 5 minutes. The fixed cells were washed three times in PBS for 5 minutes and followed by staining with 4 μg/ml DAPI for 10 minutes at room temperature. The samples were mounted on microscope slides with mounting medium and analyzed by fluorescence microscopy. The fluorescence images were processed using AutoQuant X2.1 software from Media Cybernatics, Inc. (Bethesda, USA).
Annexin V-FITC/PI apoptosis detection by flow cytometric analysis
Annexin V-FITC/PI apoptosis detection kit (Becton Dickinson, USA) was applied to apoptosis detection. Cells (3×105 per well) were seeded into 6-well plates and then treated with varying concentrations of SM as mentioned above. After 12-hour incubation, the cells were washed twice with ice-cold PBS (0.01 mmol/L, pH 7.2). Each cell sample of 100 μl were loaded and centrifuged at 200 ×g for 5 minutes. Removed the supernatant and resuspended cells in 100 μl of Annexin V/FITC binding buffer and incubated at room temperature in dark for 15 minutes with 5 μl annexin V-FITC and then added propidium iodide (PI) 10 μl (50 μg/ml) for another 5 minutes. According to the manufacturer's instructions, the apoptotic ratio was analyzed by flow cytometry (Becton Dickinson) and WinMDI 2.9 analysis software. Annexin V-positive cells were considered apoptotic.
Western blotting analysis
Bcl-2 and Bax, actin expression was investigated by Western blotting. For this purpose, MDR K562/A02 cells (2×105/ml) were exposed to different concentrations of SM at 37°C for 12 hours. Equal amounts of protein in the cell extracts were fractionated by a 12% sodium dodecyl s u l fa te (SDS)-polyac r y lamide gel and then electrotransferred to nitrocellulose membrane. Western blotting analysis was performed with primary monoclonal anti-Bcl-2, Bax, actin antibody (dilution, 1:500; Sigma, USA) using secondary biotin-conjugated goat anti-mouse IgG or anti-rabbit IgG (dilution, 1:1500; Sigma).
Quantitative analysis of P-gp
The cell-surface P-gp levels were measured by immunofluorescence flow cytometry.11 K562/A02 and K562 cells seeded into 6-well plates at a density of 2×105/ml well were treated with 3.75 μmol/L, 5.00 μmol/L and 7.50 μmol/L of SM for 12 hours, respectively. The cells were harvested, washed twice with ice-cold PBS, counted and then labeled with R-phycoerythrinconjugated mouse anti-human monoclonal antibody against P-gp according to manufacturer's instruction. The fluorescent intensity was analyzed using FACS Caliber and WinMDI 2.9 analysis software (Scripps Research Institute, USA) with isotype as control. Duplicate experiments with triplicate samples were performed.
RNA extraction and relative quantification by real-time PCR
Total RNA was extracted using the RNAeasy kit according to the manufacturer's instructions (Bioecon Biotec, China). The purity of RNA was checked by OD260/280 of RNA samples (>1.8). cDNA was synthesized through reverse transcription using M-MLV Reverse Transcriptase and Oligo (dT) primer. The MDR1 and β-actin genes expression was detected by real-time PCR assay. PCR amplification was performed in 8-tube strip format (Axygen, CA, USA) in triplicate. Each reaction contained 1×SYBR Green PCR Master mix, 1 μmol/L forward primer and reverse primer and 1 μl template cDNA in a final volume of 20 μl using a Mastercycler eprealplex apparatus (Eppendorf, Germany). Primers based on the MDR1 gene11 (sense: 5′-2;-ATATCAGCAGCCCACATCAT- 3′-2; and antisense: 5′-2;-GAAGCACTGGGATGTCCGGT- 3′-2;; 170 bp) and β-actin12 (sense: 5′-2;-AACACCCCAGCCATGTACG-3′-2; and antisense: 5′-2;-ATGTCACGCACGATTTCCC-3′-2;; 254 bp). Evaluation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, used as a control of the RNA amount, was carried out by using primer13 (sense: 5′-2;-CCATGGAGAAGGCTGGGG- 3′-2; and antisense: 5′-2;-CAAAGTTGTCATGGATGACC- 3′-2;; 195 bp). Amplification was performed for 45 cycles of sequential denaturation (95°C for 2 minutes), annealing (60°C for 15 seconds), and extension (72°C for 20 seconds). Data acquisition and the analysis of real-time PCR assay were performed using the Mastercycler ep realplex (Eppendorf, Germany). Each fluorescent reporter signal was measured against the internal reference dye signal to normalize for non-PCR-related fluorescence fluctuations between the wells. All samples were taken in triplicate independent experiments. All primers were synthesized by Sangon Co., Ltd (Shanghai, China).
Immunostaining and fluorescence microscopy
Cells were seeded onto cover slips in 24-well plates and incubated with SM for 12 hours. Cells on glass coverslips were fixed with cold methonal:acetone (1:1) for 5 minutes followed by immunostaining for actin using rabbit anti-actin antibody and phalloidin as described previously.14 The samples were mounted on microscope slides with mounting medium and analyzed by fluorescence microscopy. The fluorescence images were processed using AutoQuant X 2.1 software from Media Cybernatics, Inc. (Bethesda, USA).
All data were expressed as mean ± standard deviation (SD) and all experiments were performed at least three times. Statistical analysis was performed with an analysis of variance (ANOVA, SPSS 12.0) followed by the Turkey's t test. P values <0.05 were considered statistically significant.
Cytotoxicity of SM in MDR tumor cells
To assess the effects of SM on MDR cell lines, experiments on K562/A02, KB/VCR, and H460/Taxol, as well as their parental cell lines K562, KB, and H460 were performed to evaluate its cytotoxicity on MDR cells in vitro. Conventional anticancer drugs adriamycin, cisplatin, VCR, and Taxol, which are sensitive to MDR mechanisms, were used as the controls. SM displayed significant cytotoxicity in the examined MDR sublines, which was nearly as sensitive as or even more sensitive than the corresponding parental cell lines (Table). The resistance factors to SM of the three cell lines were far lower than those to the control drugs. Thus, the K562/A02 cells, an adriamycin-selected MDR subline that overexpresses P-gp, was selected as the model to further investigate the mechanism by which SM directly kills MDR cells.
SM-induced apoptosis in K562/A02 cells
To examine the effects of SM on the cellular morphology of MDR cells during cell death, K562/A02 cells pre-treated with SM for 12 hours were stained with DAPI (Figure 2). The SM-treated K562/A02 cells displayed dose-dependent canonical apoptotic transformation, as shown by various morphological changes including apoptotic bodies, nuclear condensation, and cell shrinkage, which were also observed under fluorescence microscopy.
To quantify further the SM-induced apoptosis in the K562/A02 cells, the cells were stained with annexin V-FITC and PI, and were subsequently analyzed by flow cytometry. The annexin V assay measures the phospholipid turnover from the inner to the outer lipid layer of the plasma membrane, an event typically associated with apoptosis. The flow cytometry analysis showed that the proportion of annexin V-staining cells was increased in the SM-treated cells (Figure 2B). After 12 hours of treatment, the percentage of annexin V-positive cells was 10.7% for the control (0.1% DMSO), and 16.89%, 32.8%, and 72.0% under SM at 3.75 μmol/L, 5.00 μmol/L, and 7.50 μmol/L, respectively. The K562/A02 cells treated with SM exhibited a significant dose-dependent increase in the number of apoptotic cells.
The Bcl-2 family plays important roles in controlling apoptosis.15 Bcl-2 and Bax proteins, two major members of the Bcl-2 family, form heterodimer complexes that cause mutual neutralization of their functions, triggering apoptosis. Therefore, the balance between the expression levels of Bcl-2 and Bax is critical in determining the fate of cells, whether survival or death. In this study, Western blotting analysis confirmed that Bcl-2 protein expression was significantly reduced and Bax was upregulated in SM-treated cells (Figure 2D).
Detection of P-gp expression by flow cytometric analysis
Overexpression of P-gp, which functions as a drug efflux pump, is one of the major mechanisms underlying MDR. A number of reports show that P-gp affects apoptosis.16,17 K562/A02 cells have been shown to express high levels of P-gp on their plasma membrane. To assess whether SM could modulate P-gp expression, the level of P-gp expression in K562/A02 and K562 cells was analyzed by flow cytometry. The cells were exposed to various concentrations of SM. The K562 cells showed virtually the same fluorescence intensity as the control when labeled with anti-P-gp monoclonal antibodies. SM-treated K562 cells expressed similar amounts of P-gp as the untreated ones (data not shown). In contrast, the K562/A02 cells exhibited a strong fluorescence peak corresponding to P-gp. After 12-hour incubation with 3.75 μmol/L, 5.00 μmol/L, and 7.50 μmol/L of SM, the expression level of P-gp was decreased by 11.2%, 38.8%, 41.7%, respectively (Figure 3A). Compared with untreated K562/A02 cells, the results revealed that SM inhibited the overexpression of P-gp, which has been well established as the cause of the MDR phenotype in many in vitro selected drug resistant cell lines. K562/A02 cells have been shown to express high levels of P-gp in their plasma membrane. Given that the P-gp is encoded by the MDR1 gene, RT-PCR analysis was carried out to assess whether SM could modulate MDR1 gene expression. The result showed that the MDR1 and actin mRNA level was decreased in the K562/A02 cells treated with SM (Figure 3B).
Effects of SM on actin expression in MDR K562/A02 and KB/VCR cells
Considering the close relationship between P-gp and cytoskeletal actin in MDR tumor cells, Western blotting and immunofluorescence staining were carried out to assess whether SM modulates actin expression. The results showed that SM exposure leads to a rapid, dramatic disruption of normal cellular architecture and actin expression was decreased in K562/A02 and KB/VCR cells treated with 3.75 μmol/L, 5.00 μmol/L, and 7.50 μmol/L SM (Figure 4).
MDR is a common major clinical problem in the treatment of cancers with conventional chemotherapeutic drugs. In our study, we demonstrated that SM possesses potent killing capacity in a panel of MDR tumor cell lines; it exhibited strong broad-spectrum cytotoxicity against MDR cells. The resistance effects and cytotoxicity of SM were superior to those of some conventional anticancer drugs, such as adriamycin, cisplatin, VCR, and Taxol in some cell lines. Its cytotoxicity against MDR tumor cells is nearly equal to or even more potent than against the corresponding parental cell lines.
Apoptosis is considered as the major process responsible for cell death in various physiological events.18,19 Examination of the morphological changes in the SM-treated cells by immunofluorescence microscopy revealed cellular features characteristic of apoptosis. Moreover, quantitative analysis of phosphatidylserine externalization through annexin V-FITC and PI staining indicated that the percentage of annexin V-positive, PI-negative cells was increased after treatment with SM, which indicated that SM induced apoptosis in K562/A02 cells. Furthermore, the expression level of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax were evaluated by western blotting. Overexpression of the anti-apoptotic molecule Bcl-2 confers resistance to anticancer treatment.20 These results showed that SM upregulated Bax but downregulated Bcl-2 protein expression, which provided a possible explanation for the observed apoptosis in the experiment.
Evidence have demonstrated that functional P-gp confers resistance to apoptosis induced by diverse nondrug stimuli, including Fas, TNF, UVB-irradiation, γ-irradiation and serum starvation.21,22 Moreover, some oncogenes (e.g., Bcl-2) that are involved in apoptotic mechanisms subsequently activate anti-apoptotic pathways.23 In our study, SM inhibited P-gp and MDR1 gene expression. These data indicated that the apoptosis induced by SM might relate to its inhibition of P-gp in MDR cells lines. Moreover, the reduction of MDR1 expression by SM possibly contributes to its cytotoxicity and apoptotic induction in this system.
The cytoskeleton has been shown to be involved in apoptosis and the expression of P-gp.24 Increasing evidences have indicated a close relationship between P-gp and actin in the cytoskeleton of MDR tumor cells.25 By observing the effect of SM on the morphology of actin through immunofluorescence analysis of KB/VCR and K562/A02 cells, facilitating observation of the cross-linked actin filaments, we found that the microfilament network was destroyed in KB/VCR and K562/A02 cells pretreated with SM. These results suggest that the P-gp-actin association pathway might be involved in SM-induced apoptosis in MDR cells.
However, our data indicate that SM at non-cytotoxic doses had no effect on decreasing P-gp activity, as well as protein and gene expression, which was consistent with the results of the MTT assay (data not shown), indicating that SM at non-cytotoxic doses had no reversal effect on MDR K562/A02 cells. On the other hand, SM at concentrations that induce P-gp downregulation triggered cytotoxicity and apoptosis in MDR K562/A02 cells. This action differs from those of a variety of reversal agents, such as verapamil and CsA. Thus, SM is a novel chemotherapy agent bypassing MDR mechanisms, which may provides a new insight into alternative ways to overcome MDR in cancer chemotherapy.
Taken together, these findings suggest that SM has broad-spectrum cytotoxic activity against multiple MDR cell lines. The imbalance between the Bax and Bcl-2 contributes to SM-induced apoptosis in MDR K562/A02 cells. SM decreases P-gp expression and destroys the microfilament network. However, the mechanism by which SM affects the P-gp-actin pathway remains unclear and further studies are warranted. Collectively, these data suggest that SM is a promising natural pro-apoptotic agent that bypasses the MDR mechanism in MDR tumor therapy.
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