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
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
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
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.
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
[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]
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.
Our study was supported, in part, by an USAMRMC Project Grant
(Contract #W81XWH-07-2-0078), University Research Grant FY 2002-2009 and
Bhattacharya, R., Mukherjee, P. (2008) Biological properties of
"naked" metal nanoparticles. Adv. Drug Deliv. Rev. 60(11),
Bhushan, A., Hacker, M.P., Tritton T.R. (1999) Collateral
methotrexate resistance in cisplatin-selected murine leukemia cells.
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Calsou, P, Sailes, B. (1993) Role of DNA repair in the mechanisms
of cell resistance to alkylating agents and cisplatin. Cancer Chemother.
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Giaccone, G., Pinedo H.M. (1996) Drug resistance. Oncologist,
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Jordan, P., Carmo-Fonseca, M. (2000) Cell Mol. Life Sci. Molecular
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Kartalou, M., Essigmann, J.M. (2001) Mechanisms of resistance to
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Lai, J. C., Lai, M.B., Jandhyam, S., Dukhande, V.V, Bhushan, A.,
Daniels,C.K., Leung, S.W. (2008) Exposure to titanium dioxide and other
metallic oxide nanoparticles induces cytotoxicity on human neural cells
and fibroblasts. Int. J. Nanomedicine, 3(4), 533-545.
Liu, F.S. (2009) Mechanisms of chemotherapeutic drug resistance in
cancer therapy-A quick review Taiwan J. Obstet. Gynecol. 48(3), 239-244.
Siddik, Z.H. (2003) Cisplatin: mode of cytotoxic action and
molecular basis of resistance. Oncogene, 22(47), 7265-79.
Welters, M.J., Fichtinger-Schepman, A.M., Baan, R.A., Flens, M.J.,
Scheper, R.J., Braakhuis, B.J. (1998) Role of glutathione, glutathione
S-transferases and multidrug resistance-related proteins in cisplatin
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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:
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:firstname.lastname@example.org