Background: There are two general categories
of drug resistance: acquired and intrinsic. The mechanisms involved
in acquired drug resistance have been extensively studied, and several
mechanisms have been described. However, the mechanisms responsible
for intrinsic drug resistance have not been elucidated, to our knowledge.
The purpose of the present study was to investigate the cytological and
biochemical differences between acquired and intrinsic drug resistance
in osteosarcoma cells.
Methods: We previously isolated a clonal cell
line (MOS/ADR1) to study acquired resistance in osteosarcoma by
exposure of parental murine osteosarcoma cells (MOS) to doxorubicin.
In the present study, we cloned a new, intrinsically resistant cell
line (MOS/IR1) by single-cell culture of MOS cells and we investigated
the differences in cell phenotype and the mechanisms of resistance
in both of these resistant clones.
Results: The MOS/ADR1 and MOS/IR1 cells were
sevenfold and fivefold more resistant to doxorubicin than the parental
murine osteosarcoma cells. Morphologically, the MOS/ADR1 cell line
was composed of polygonal cells, whereas the MOS/IR1 cell line consisted
of plump spindle cells with long cytoplasmic processes. The MOS/IR1
cells showed a much lower level of alkaline phosphatase activity
than did the MOS/ADR1 and MOS cells. There were no substantial differences in
the cellular DNA content or the doubling time among these three
Overexpression of the P-glycoprotein involved in the function
of an energy-dependent drug-efflux pump was detected in the MOS/ADR1
cells but not in the MOS/IR1 cells. After the cells were incubated
with doxorubicin for one hour, the two resistant lines had less
accumulation of the drug than did the parent line (p < 0.05).
The addition of a P-glycoprotein antagonist, verapamil, or the depletion
of cellular adenosine triphosphate resulted in a marked increase
in the accumulation of doxorubicin in the MOS/ADR1 cells (p < 0.05)
but not in the MOS/IR1 cells. The MOS/ADR1 cells were found to exhibit
cross-resistance only to substrates for P-glycoprotein (such as
doxorubicin, vincristine, and etoposide), whereas the MOS/IR1 cells were
resistant to all of the drugs studied (including cisplatin and methotrexate).
The degree of drug resistance in the MOS/IR1 cells was found
to be associated with the molecular weight of the drugs (p < 0.05).
Permeabilization of the plasma membrane by saponin increased both
the accumulation of doxorubicin (p < 0.05) and the cytotoxic
activity of this drug in all lines, but the effects were most pronounced
in the MOS/IR1 cells.
Conclusions: Taken together, this data suggests
that reduced drug accumulation in the MOS/IR1 cells may be due to
the effect of decreased permeability of the plasma membrane on the
transport of drugs from the extracellular environment into the cytosol
of the cell and that this may be the mechanism responsible for intrinsic
resistance to multiple drugs in the MOS/IR1 cell line.
Clinical Relevance: Current drug treatment for
human osteosarcoma may include multiple chemotherapeutic agents, such
as doxorubicin, cisplatin, and methotrexate. These drugs exhibit
different cytotoxic actions and, thus, the mechanisms of resistance
to individual drugs vary. Clinical resistance to multidrug chemotherapy
may be observed in tumors that recur after repetitive chemotherapy
and in previously untreated tumors. In the former group, a tumor cell
may express multidrug resistance by combining several different
mechanisms due to its exposure to various drugs. In the latter group,
however, this is not likely. Decreased intracellular drug accumulation
due to reduced permeability of the plasma membrane, found in the
MOS/IR1 cells, is one possible mechanism and may explain the intrinsic
resistance to multidrug chemotherapy for the treatment of osteosarcoma.
Further study regarding the resistance mechanism in the MOS/IR1 cells
may help to overcome the intrinsic drug resistance in osteosarcoma.