Abstract
Background/Aim: Pyrimethamine (PYR), an anti-malarial drug is known to inhibit various types of human cancer cells. The aim of this study was to investigate the anti-tumour effects of pyrimethamine (PYR) and its underlying molecular mechanisms using the human NSCLC cell line A549. Materials and Methods: PYR was dissolved in dimethyl sulfoxide to determine its apoptotic activity on A549 cells. Cell viability was determined by the MTT assay. Cell cycle, mitochondrial membrane potential, and Annexin V-FITC early apoptosis detection were evaluated by flow cytometry. Cyclin-dependent kinase (CDK) and Bcl-2 family protein expression was determined by western blotting. Results: PYR reduced cell viability percentage and induced G0/G1 arrest, which was associated with down-regulation of cyclins D1 and E, CDK4, and CDK2, and up-regulation of p21. PYR induced sub-G1 accumulation, Annexin-V binding, caspase-9 and -3 activation, poly (ADPribose) polymerase cleavage, and mitochondrial dysfunction in A549 cells. Moreover, PYR effectively inhibited NSCLC tumour growth in an A549 xenograft model. Conclusion: PYR demonstrated anti-tumour effects on NSCLC in vitro and in vivo, indicating its therapeutic potential against human NSCLC.
The incidence of lung cancer continues to rise and has become the leading cause of cancer deaths worldwide (1). Two major types, non-small cell lung cancers (NSCLCs) and small-cell lung cancers (SCLCs) can be identified. NSCLCs account for approximately 80% of all cases. Surgery is regarded the primary treatment modality for NSCLCs, but only 20-25% of the tumors are suitable for potentially curative resection. As a result, more effective treatments, such as chemotherapy (CT) or chemotherapy concurrent with radiotherapy (RT) to improve recurrence and mortality rates, are needed (2). Different chemotherapeutic agents have been used for NSCLCs to improve prognosis. However, NSCLCs are relatively resistant to chemotherapy, and the 5-year survival rate is as low as 20% (3). Therefore, there is an urgent need to identify or develop new agents to improve sensitivity or resistance to current chemotherapy in the treatment of NSCLC.
Despite intensive research, improvements in the treatment of NSCLCs have remained deficient. Currently, the chemotherapy use for NSCLCs often consists of a combination of drugs, the most commonly used being cisplatin-based plus docetaxel (Taxotere), gemcitabine (Gemzar), paclitaxel (Taxol and others), vinorelbine (Navelbine and others) and so on (4). These cytotoxic drugs, irrespective of their intracellular target, have been shown to cause cell death in sensitive cells, at least partly, by inducing apoptosis. Apoptosis, a process of programmed cell death, could be induced by activation of caspases in the extrinsic pathway or through mitochondrial damage and apoptotic protein activation in the intrinsic pathway. Therefore, different biochemical targets or agents that play a role in intrinsic and extrinsic apoptotic pathways should be investigated for cancer treatment (5-8).
Pyrimethamine (PYR), a folic acid antagonist, can indirectly block the synthesis of nucleic acids by inhibiting the enzyme dihydrofolate reductase (DHFR) (9). Currently, it is effectively used in the prevention and treatment of opportunistic infections such as malaria and toxoplasmosis (10, 11). Several investigators have noted that PYR has antitumor activity. Giammarioli et al. indicated that PYR induced apoptosis through caspase and cathepsin mechanisms in melanoma cells (12). PYR is also reported to enhance temozolomide-induced cytotoxicity in melanoma cells and invasive pituitary adenomas (13, 14). Moreover, PYR is known to sensitize temozolomide-resistant glioblastoma and act as a selective inhibitor in acute myeloid leukemia (15, 16). Furthermore, PYR also suppressed the telomerase activity in the PC-3 prostate cancer cell line (17) and inhibited the matrix metalloproteinases (MMP) type 2 activity in fibrosarcoma (18). However, no data on the role of PYR in NSCLC cells are presently available or extensively studied.
Therefore, in this study, the in vitro and in vivo antitumor activity of PYR was studied in the human NSCLC cell line A549. In addition, the possible molecular mechanisms responsible for its anticancer activity were also investigated.
Materials and Methods
Cell lines and culture conditions. The NSCLC cell line A549 was purchased from the American Type Culture Collection (ATCC®) (Manassas, VA, USA). The human NSCLC cell line A549 was maintained in RPMI with 10% fetal bovine serum, 1% penicillin and streptomycin (Gibco/BRL, Gran Island, NY, USA) and was cultured at 5% CO2 and saturated humidity at 37°C in an incubator.
Cell viability assay. The A549 cells were seeded at 1×104 in a 24-well dish overnight. Pyrimethamine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at 100 mM as stock solution for in vitro assays. The cells were treated with 25, 50 and 100 μM for 24 h, 48 h and 72 h. Then, the cells were cultured with 1 mg/ml 3-(4,5-Cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (BioVision, Milpitas, CA, USA) for 4 h, after which the medium was removed. The wells were washed once with Phosphate-buffered saline (PBS), and formazan was dissolved in DMSO. The solution was measured by microplate reader (Tecan Sunrise, San Jose, CA, USA) at 540 nm.
Cell-cycle assay. The A549 cells were seeded at 1×105 in a 6-well dish overnight. The cells were treated with 25, 50 and 100 μM pyrimethamine for 24, 48 and 72 h. The cells were collected and washed with PBS, and then, the cells were fixed in 70% ethanol and kept at −20°C for 16 h or overnight. The cells were washed with PBS and stained with propidium iodide (PI) solution (containing 2 mg/ml RNase, 1 mg/ml PI and 5% Triton X-100) (Sigma-Aldrich) for 30 min at RT and kept away from light. Fluorescence intensity in all samples was detected and measured using an Accuri™ C5 cytometer (BD Biosciences, San Jose, CA, USA).
Annexin V-FITC/PI staining assay. The A549 cells were seeded at 1×105 in a 6-well dish overnight. The cells were treated with 25, 50 and 100 μM pyrimethamine for 24, 48 and 72 h. The cells were collected and washed with PBS and stained with Annexin-V/PI solution (containing 2 μl Annexin V, 2 μl PI and 100 μl binding buffer) (BioVision, Milpitas, CA, USA) for 15 min at RT and kept away from light. The fluorescence intensity of all samples was detected and measured using an Accuri™ C5 cytometer (BD Biosciences).
Caspase-3, 8, and 9 activities Assay. After treatment with PYR for 48 h, cells were harvested for examination of caspase-3, 8, and 9 activities using the appropriate CaspGLOW™ Fluorescein Active Caspase Staining Kits (Biovision, Milpitas, CA, USA) according to the manufacturer's specifications.
Mitochondrial membrane potential detection. The A549 cells were seeded at 1×105 in a 6-well dish overnight. The cells were treated with 25, 50 and 100 μM pyrimethamine for 24, 48 and 72 h. The cells were collected, washed with PBS and stained with 10 μg/ml JC-1 (Life Invitrogen, Carlsbad, CA, USA) for 10 min at RT and kept away from light. The fluorescence intensity of all samples was detected and measured using an Accuri™ C5 cytometer (BD Biosciences).
Cytochrome c level assay. The A549 cells were seeded at 1×105 in a 6-well dish overnight. The cells were treated with 25, 50 and 100 μM pyrimethamine for 24 h, 48 h and 72 h. Cells were then collected, washed with PBS and the cell membrane was permeabilized with 1 mg/ml digitonin buffer for 5 min in ice and washed with PBS containing 0.1% BSA. The samples were fixed and processed with a Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer's instructions. Next, the samples were incubated with cytochrome c-FITC antibody for 1 h in ice and kept away from light. The fluorescence intensity of all samples was detected and measured using an Accuri™ C5 cytometer (BD Biosciences).
Western blotting. The A549 cells were seeded at 1×106 in a 10-cm dish overnight. The cells were treated with 25, 50 and 100 μM pyrimethamine for 24 h and collected to analyze the level of cell cycle-associated protein and apoptotic-related protein. The cells were lysed in RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor, and protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Thirty micrograms of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was incubated with primary antibody at 4°C overnight. After that, the membrane was incubated with peroxidase-labeled secondary antibody for 4°C overnight. The immunofluorescence signals were detected with an enhanced chemiluminescence (ECL) system and developed using the LAS3000 system (Fujifilm, Valhalla, NY, USA). Densitometric analysis performed in this study was conducted using Image J software (National Institute of Health, Bethesda, MD, USA). In this study, the primary antibodies were anti-BCL-xL (1:1,000 dilution; clone S-18, CAS:sc-634, Santa Cruz, St. Louis, MO, USA), anti-Bak (1:1,000 dilution;, CAS:sc-832, Santa Cruz), anti-BCL-2 (1:1,000 dilution; clone 50E3, CAS:2870, Cell Signaling, Beverly, MA, USA), anti-CDK2 (1:5,000 dilution; clone E304, catalog:1134-5, Epitomics, Cambridge, MA, USA), anti-CDK4 (1:1,000 dilution; clone polyclonal, catalog:S1572, Epitomics), anti-Cyclin D1 (1:1,0000 dilution; clone EPR2241(IHC)-32, catalog:2261-5, Epitomics), anti-Cyclin E (1:1,000 dilution; clone D7T3U, CAS:20808, Cell Signaling), anti-p21 (1:10,000 dilution; clone EP1125Y, catalog:ab52939, Abcam, Cambridge, MA, USA), and anti-β-actin (1:1,000;clone C4, CAS:sc-47778, Santa Cruz). The blots were quantified by densitometric analysis using Image J software version 1.47 (National Institutes of Health, Bethesda, MD, USA).
Animal. Female BALB/c nu/nu mice (n=24; 4-weeks-old; 20-25 g) were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The mice were housed in cages with free access to food and water at controlled temperature (22±2°C) and humidity (45-65%) on a 12-h light/dark cycle. All animal procedures were conducted according to institutional guidelines and approved by the Institutional Animal Care and Utilization Committee of National Chung Hsing University, Taiwan (approval protocol no. NCHU-IACUC NO. 105-104).
Xenograft animal model. Quantities of 1×107 A549 cells were injected into the right flanks of 6-week-old female BALB/c nu/nu mice. When the tumors were established (day 10), the mice were randomly divided into two groups: the vehicle control group was given 10% DMSO + 90% glyceryl trioctanoate (Sigma-Aldrich), while the PYR treatment group received 30 mg/kg of PYR via intraperitoneal injection (i.p.) once a day. The tumor volume was measured once every five days. On day 35, mice were sacrificed by gradual-fill 100% CO2 inhalation (rate of 30-40% of the chamber volume per minute) and tumors were extracted, photographed and weighed. The tumor volume was measured by the following formula, length × width × width/2, and averaged (mean±standard error of mean).
Statistical analyses. Statistical analyses and graphics were performed using GraphPad Prism Version 5.0 (San Diego, CA, USA). The Mann–Whitney U-test was used to compare the two groups based on the animal experiments. For comparison between the control and drug-treatment groups, the two-tailed t-test (Student's t-test) was used. A p-value less than 0.05 was considered statistically significant.
Results
PYR inhibits cell proliferation through G1 phase arrest. First, we evaluated the dose-dependent and time-dependent effect of PYR on the NSCLC A549 cell line. As shown in Figure 1A, PYR can gradually reduce the cell viability of A549 with higher concentrations and longer times. In addition, microscopic examination revealed that morphological changes in cell shape such as shrinkage, rounding, and floating of the cells were observed with increasing concentrations of PYR (Figure 1B). To further assess the effect of PYR on cell proliferation, we tested the fluorescence intensity by flow cytometry during different cell cycles. For the A549 cell line, PYR induced G1 and sub-G1 phase accumulation of cells after 24 h treatment, as shown in Figure 1C. In addition, the number of cells at the sub-G1 phase increased at different PYR concentrations (25, 50, 100 μM) (Figure 1C) at 48 h. The western blotting results also showed that incubation with PYR for 24 h reduced the expression of G1 phase-related proteins, including CDK4, CDK2, cyclin E and cyclin D1; however, the expression of Cdk2 inhibitor p21Cip1 was increased (Figure 1D). These results suggest that PYR can inhibit cell proliferation via G1 phase arrest.
PYR promotes apoptosis by inducing apoptotic protein activation. Next, the effect of PYR on apoptosis of A549 cells was investigated. Previous literature has reported that apoptotic cells express phosphatidylserine (PS) on the surface (19, 20). In this study, PYR stimulated PS expression, as shown by flow cytometry using Annexin V-FITC staining. As depicted in Figure 2A, PYR dose-dependently induced PS expression after 48 h. The results of the caspase activity assay also revealed that PYR significantly increased caspase-9 and caspase-3 activities at 48 h but not caspase-8 activity (Figure 2B). In addition, western blotting results showed that the mitochondrial membrane pro-apoptotic protein Bak was increased and anti-apoptotic proteins Bcl-xL and BCl-2 were decreased (Figure 2C). The alteration of Bak and Bcl-2 levels resulted in an increase in the ratio of Bak to Bcl-2 (Figure 2D). Furthermore, PYR induced sub-G1 phase accumulation at 48 h, as shown in Figure 1B. These results indicated that PYR inhibits cell proliferation via activation of apoptosis.
PYR induces apoptosis via mitochondrial dysfunction and cytochrome c release. Due to the increased Bak and decreased BCl-2 and Bcl-xL levels found in western blotting, as well as the cleavaged-caspase-8 not activated by PYR, it is evident that PYR may cause cancer cell apoptosis via the mitochondrial (intrinsic) apoptotic pathway. Mitochondrial membrane potential (MMP) analysis revealed that JC-1, an indicator for MMP, forms monomers (green fluorescence) in low-MMP conditions and JC1-aggregates (red fluorescence) in normal conditions. As shown in Figure 3A, PYR can reduce the mitochondrial membrane potential by inducing green fluorescence accumulation at 48 h. This reduced mitochondrial membrane potential also enhances the cytochrome c release into the cytosol, as shown in Figure 3B. These results suggest that PYR can induce apoptosis via mitochondrial dysfunction.
PYR suppresses tumor growth in xenograft animal model. In a xenograft animal model, it was observed that PYR inhibited tumor growth in tumor-bearing mice. On day 10, the PYR (30 mg/kg)-injected mice group revealed a reduction in tumor growth, as shown in Figure 4A. After these mice were sacrificed on day 35, the PYR-treated tumor size was significantly smaller, and the average weight was also lighter than the vehicle control group (Figure 4B-C). These results supported that PYR could suppress tumor growth in vivo.
Discussion
This is the first study to determine the in vitro and in vivo antitumor activity of PYR in human NSCLC cells. In the present work, our data showed that PYR promotes the death of cancer cells through G1 cell-cycle arrest and the intrinsic (mitochondrial mediated) apoptotic pathway in the NSCLC cell line. The in vivo antitumor effect of PYR was also observed in a severe SCID-mouse xenograft model experiment.
Lung cancer is common worldwide, with an estimated 1.56 million deaths annually (1). Patients can receive combined modality with surgery, chemotherapy and radiotherapy for advanced disease, but the outcome has remained stagnant in the past several decades (2). Recently, PYR, which is an anti-folate drug, has been studied as an antitumor agent or chemotherapy enhancer (13, 17). However, its antitumor effect for lung cancer is so far unknown. Previous reports have indicated that PYR could induce apoptosis and S-phase accumulation in many cancer cell lines (12, 21, 22). However, in this study PYR induced the G0/G1 phase arrest and increased sub-G1 phase accumulation. The results of western blotting also showed that the levels of cyclin E, cyclin D1, CDK4 and CDK2 were reduced, but p21 was increased. These results suggested that PYR inhibits NSCLC proliferation by incomplete DNA synthesis and cell division.
Although cell death can occur through non-apoptotic mechanisms, apoptosis is the major mechanism of action of chemotherapeutic agents against cancer cells (23, 24). Thus, induction of apoptosis is considered to be a potent therapeutic strategy in the eradication of tumors, especially lung malignance (25, 26). To test this, we analyzed the activity of caspases 3, 8, and 9. Caspase-3 is the most important component of the apoptotic pathway and can be activated by caspase-8 or -9, the two key proteins of the extrinsic and intrinsic apoptotic pathways, respectively. The mechanism underlying PYR-induced apoptosis has been investigated for years. Dai et al. reported that PYR caused a significant increase in the activities of caspase 3/7, 8, 9 in melanoma cell lines (14). Consistent with another previous report (12), the apoptotic proteins caspase-9 and -3, but not caspase-8, were activated in our study, indicating that PYR exerts its effects via the intrinsic apoptotic pathway.
Because PYR induced apoptosis through the intrinsic pathway (mitochondrial damage), we further attempted to analyze the related pathway. If the mitochondria were damaged, the mitochondria membrane potential (MMP) would be decreased at the same time. In mitochondrial membrane potential (MMP) analysis, JC-1, an indicator for MMP, showed a reduced expression of green fluorescence accumulation. Also, flow cytometry analysis of cytochrome c-FITC staining indicated that PYR induced mitochondria to release cytochrome c into the cytoplasm. Apoptosis regulator Bcl-2 family proteins, either pro-apoptotic (such as Bax, BAD, Bak) or anti-apoptotic (Bcl-2 proper, Bcl-xL), are the two major types that regulate mitochondria-related apoptosis (27). In our results, the levels of Bcl-2 and Bcl-xL were reduced, but Bak was significantly increased at 48 h (Figure 2C). These results suggest that PYR induces apoptosis through the mitochondria-damaged pathway.
In conclusion, the results of the present study showed that PYR promoted the death of cancer cells through G1 cell-cycle arrest and the intrinsic (mitochondrial mediated) apoptotic pathway in the NSCLC cell line. Inhibition of cell proliferation suggests that PYR could be used for anti-cancer therapy. Further study is needed to evaluate its efficacy when combined with current chemotherapy regimens.
Acknowledgements
The Authors thank the reviewers for their helpful comments on this article and the present study was supported by Changhua Christian Hospital (grant nos. 103-CCH-IRP-003 and 103-CCH-ICO-001).
Footnotes
Conflicts of Interest
The Authors have no conflict of interest associated with the manuscript.
- Received March 27, 2018.
- Revision received April 27, 2018.
- Accepted April 30, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved