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Differential cytotoxic effects of magnesium oxide nanoparticles on cisplatin-sensitive and cisplatin-resistant leukemia cancer cells.
Article Type:
Report
Subject:
Magnesium oxide (Chemical properties)
Magnesium oxide (Health aspects)
Magnesium oxide (Atomic properties)
Nanoparticles (Chemical properties)
Nanoparticles (Health aspects)
Nanoparticles (Composition)
Leukemia (Health aspects)
Cancer cells (Chemical properties)
Cancer cells (Health aspects)
Authors:
Patil, Prathamesh P.
Lai, Maria B.
Leung, Solomon W.
Lai, James C.K.
Bhushan, Alok
Pub Date:
06/01/2010
Publication:
Name: Journal of the Idaho Academy of Science Publisher: Idaho Academy of Science Audience: Academic Format: Magazine/Journal Subject: Science and technology Copyright: COPYRIGHT 2010 Idaho Academy of Science ISSN: 0536-3012
Issue:
Date: June, 2010 Source Volume: 46 Source Issue: 1
Product:
Product Code: 2819849 Magnesium Oxide NAICS Code: 325188 All Other Basic Inorganic Chemical Manufacturing SIC Code: 2819 Industrial inorganic chemicals, not elsewhere classified
Geographic:
Geographic Scope: United States Geographic Code: 1USA United States

Accession Number:
240693401
Full Text:
ABSTRACT

Magnesium oxide (MgO) has many applications in pharmaceutical, cosmetic and food industry. It has been extensively used in these industries for decades. Magnesium oxide is used in preparation of antacid, laxatives and mineral supplements. It is also used in cosmetics in preparation of sunscreens, toothpastes and dental cements. In previous studies, we have characterized the effects of different metal oxide nanoparticles on normal fibroblast cells and cancer cells. In this study, we compared the cytotoxicity of magnesium oxide nanoparticles on routine leukemia cells (L1210) and cisplatin-resistant sub-line (L1210/DDP). We found that magnesium oxide nanoparticles exhibit differential cytotoxicity in L1210 and L1210/DDP leukemia cells. Our results also suggested that cisplatin-resistant cancer cells were cross-resistant to MgO nanoparticles. These findings may assume pathophysiologic importance in elucidation of mechanisms underlying anti-cancer drug resistance and may aid future efforts in developing novel anti-cancer drugs.

Key Words: cancer, resistance, nanoparticles, leukemia

INTRODUCTION

Approaches to treat cancers include surgery, chemotherapy and radiation therapy. Conventional chemotherapeutic agents like cisplatin, methotrexate, and 5-flurouracil are used extensively in the treatment of a variety of cancers. One major reason for the treatment failure is resistance to primary chemotherapeutic agents (Gottesman et al. 2002). Certain cancers which develop resistance to primary cancer chemotherapeutic agent(s) also develop cross-resistance to other unrelated chemotherapeutic agents (Bhushan et al. 1999).

Several mechanisms that contribute to the development of resistance in cancers have been proposed. Some of these mechanisms include increased efflux of the drug out of the cancer cell, decreased influx of the drug into the cancer cell, and increase in altered cellular targets (Siddik et al. 2003, Liu et al. 2009). Other studies implicate the involvement of altered cellular signaling in the development of resistance to chemotherapy in cancer cells (Jordan et al. 2000).

Development of resistance in cancer cells is the major cause of failure of chemotherapy in treatment of cancer (Giaccone et al. 1996). Thus, overcoming drug resistance is a major challenge in the effective use of chemotherapeutic drugs to treat cancers. Better understanding of the mechanisms that contribute to drug resistance is necessary so that novel strategies can be devised for more effective treatment of cancer.

Metallic nanoparticles are a subtype of nanoparticles made from metals and their compounds (e.g., titanium dioxide, zinc oxide, MgO, gold, silver). The use of nanomaterials enables new technologies and approaches in the development of drugs for the treatment of diseases. Owing to their small size, metallic nanoparticles differ from their bulk counterparts in several properties like magnetic, surface electric, and optical. These properties make metallic nanoparticles attractive candidates for developing agents for diagnostic, imaging, and therapeutic purposes (Bhattacharya et al. 2008).

Work in our laboratory focuses on the biological and toxicological effects of nanomaterials. In previous studies, we have examined the effects of different metallic nanoparticles on neural tumor cells and normal human fibroblasts. We found that zinc oxide and titanium oxide nanoparticles induced high cytotoxicity in normal fibroblasts, while magnesium oxide nanoparticles exhibited comparatively low cytotoxicity on these cells even at high concentrations (Lai et al. 2008). Because MgO nanoparticles exhibited low cytotoxicity on normal fibroblasts, we selected them for further studies to determine whether they could be used to overcome drug resistance in cisplatin-resistant leukemia cancer cells.

Materials and Methods: MgO nanopowder (Sigma-Aldrich Cat no: 549649, 50 nm particle size nanopowder) was dispersed in 100 ml of sterile saline in a sealed conical flask and stirred at ambient temperature for four hours prior to be diluted to the specified concentrations for treatment of cells. McCoy's 5A medium was purchased from Sigma-Aldrich. Mouse polyclonal antibodies for Akt and Erk, respectively, were obtained from Santa Cruz Biotechnology.

Cells and Culture Conditions: L1210 murine leukemia cells were purchased from Sigma-Aldrich and maintained under sterile conditions at 37[degrees]C and 5 % C[O.sub.2] in McCoy's 5A mediumsupplemented with 10% (v/v) horse serum. The L1210/ DDP murine leukemia cells were obtained as a subline of L1210 cells resistant to cisplatin and were maintained under sterile conditions at 37[degrees]C and 5% C[O.sub.2] in McCoy's 5A mediumsupplemented with 10% (v/v) fetal bovine serum.

Dose Response Assay: L1210 and L1210/DDP cells were split at equal density in each well of 24-well plates. Cells in each well were treated with specified concentrations of MgO nanoparticles (ranging from 0.1 [micro]g/ml to 100 [micro]g/ml) for 72 hours at 37[degrees]C. After 72 hours, an aliquot was used for counting using a Z2 Beckman coulter counter.

Western Blot Analysis: L1210 cells were treated with various concentrations of MgO nanoparticles (10, 25, 50, or 100 [micro]g/ml) for 72 hours. After treating the cells with MgO nanoparticles, cell lysates were prepared using the lysis buffer (1% (v/v) Triron X-100, 10 mM Tris base pH 7.6, 5 mM EDTA, 50 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM sofium fluoride, 0.1% (w/v) sodium azide, 50 mM phenyl methyl sulphonyl fluoride, 0.5 mg/ml aprotinin, 2.5 mg/ml leupeptin, and 100 mM sodium orthovanadate in distilled water, pH 7.6). BioRad reagents (Bradford assay) were used to determine the protein concentration in cell lysates. 25 pg of protein sample was loaded onto the wells in the sodium dodecyl sulphate polyacrylamide gel. The proteins separated by gel electrolysis were transferred to polyvinylidene fluoride membrane (PVDF) and blocked with the blocking solution (5% (w/v) Tris buffered saline with 0.01% (v/v) Tween or TBST in no-fat powdered milk). After washing, the blots were incubated in 1:500 primary antibody solution prepared in 5% bovine serum albumin (BSA) in distilled water. Membranes were washed and treated with the secondary antibody and developed using chemiluminescence kit (Pierce biotechnology, Rockford, Illinois), as recommended by the manufacturer. The blots were analyzed for the levels of Akt, pAkt, Erk and pErk. The scans were digitized using Unscan-it-gel 6.1 software.

RESULTS

Dose Response Studies: L1210 and L1210/DDP cells were treated with different concentrations of magnesium oxide nanoparticles for 72 hours. The dose-response studies were performed following the procedure described above. The results revealed that at the concentrations employed, MgO nanoparticles did not exert any cytotoxic effect on L1210/DDP cells after 72 hours treatment (Figure 1). By contrast, MgO nanoparticles did exert some cytotoxic effects on L1210 cells after 72 hours treatment (Figure 1). When the effects of MgO nanoparticles on the two cell lines were compared (Figure 1), it was evident that the resistant L1210/DDP cells were not susceptible to the cytotoxicity of the nanoparticles at the concentrations used while the sensitive L1210 cells showed dose-related decreases in survival upon treatment with the nanoparticles.

[FIGURE 1 OMITTED]

Akt and Erk expression: We performed western blot analysis to assess the change in expression of cell signaling elements like Akt, pAkt, Erk and pErk in L1210 cells after MgO nanoparticles treatment using a procedure described above. Our results showed that the expression of pAkt and pErk in these cells decreased when treated with increasing concentrations of MgO nanoparticles (Figure 2).

[FIGURE 2 OMITTED]

DISCUSSION

A major reason for the treatment failure in a number of cancers is the development of resistance to chemotherapy. Some of the proposed mechanisms that contribute to drug resistance for cisplatin include increase in intracellular glutathione, increase in MRP protein expression, and increase in DNA repair mechanisms (Welters et al. 1998, Calsou et al. 1993). Despite these suggested mechanisms, a definitive mechanism for cisplatin resistance remains to be elucidated. In our laboratory, we are developing a novel approach towards using metal-based nanoparticles in overcoming cancer drug resistance.

Although magnesium oxide nanoparticles did not overcome drug resistance in L1210/DDP cells, our results from the dose-response studies showed that the cisplatin-resistant L1210/DDP leukemia cancer cells are cross-resistant to MgO nanoparticles. We also observed that cisplatin-sensitive leukemia cells are sensitive to MgO nanoparticles. These observations suggest that L1210/DDP may have similar mechanisms of drug resistance for cisplatin and for MgO nanoparticles.

Altered expression of signal transduction pathways are known to play a role in drug resistance. Our analysis of cell signaling proteins like Akt, pAkt, Frk and pErk by western blot analysis showed that the MgO nanoparticles decreased the expression of phosphorylated forms of Akt and Erk in the sensitive cell line. The decrease in the levels of phosphorylated forms of Akt and Erk with increasing concentrations of MgO nanoparticles in L1210 cells is highly suggestive of down-regulation of pathways leading to cellular proliferation and other processes critical for cancer progression. Clearly, this is an important area that merits further investigation.

ACKNOWLEDGEMENTS

Our study was supported, in part, by an USAMRMC Project Grant (Contract #W81XWH-07-2-0078), University Research Grant FY 2002-2009 and NIH P20RR016454.

REFERENCES

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Prathamesh P. Patil (1,3), Maria B. Lai (1,3), Solomon W. Leung (2,3), James C.K. Lai (1,3), and Alok Bhushan (1,3)

(1) Department of Biomedical & Pharmaceutical Sciences, College of Pharmacy; (2) Department of Civil and Environmental Engineering, College of Engineering; and (3) Biomedical Research Institute, Idaho State University, Pocatello, ID 83209-8334

Address for correspondence:

Alok Bhushan

Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, Pocatello, ID 83209-8334 Phone: 208-282-4408 * Fax: 208-282-4482 Email:abhushan@pharmacy.isu.edu
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