Regulation of Multidrug Resistance in Cancer Cells by Hyaluronan*
Suniti Misra,
Shibnath Ghatak,
Alexandra Zoltan-Jones and
Bryan P. Toole
From the
Department of Anatomy and Cellular Biology, Tufts University School of
Medicine, Boston, Massachusetts 02111
Received for publication, April 22, 2003
, and in revised form, April 29, 2003.
 |
ABSTRACT
|
---|
Multidrug resistance in cancer cells is often due to ATP-dependent efflux
pumps, but is also linked to alterations in cell survival and apoptotic
signaling pathways. We have found previously that perturbation of
hyaluronan-tumor cell interaction by treatment with hyaluronan
oligosaccharides suppresses the phosphoinositide 3-kinase/Akt cell survival
signaling pathway in cancer cells and reduces tumor growth in vivo.
Here we find that these oligomers suppress both the MAP kinase and
phosphoinositide 3-kinase pathways in multidrug resistant tumor cells and
sensitize these cells to a variety of chemotherapeutic drugs. On the other
hand, increased hyaluronan production induces resistance in drug-sensitive
tumor cells. Likewise, increased expression of emmprin, which is a
glycoprotein that is present on the surface of most malignant cancer cells and
that stimulates hyaluronan production, also induces increased resistance.
Thus, perturbation of hyaluronan signaling may provide a dual therapeutic
role, since it has intrinsic suppressive effects on tumor growth as well as
sensitizing cancer cells to chemotherapeutic agents.
 |
INTRODUCTION
|
---|
Multidrug resistance in cancer arises by several mechanisms, among which
are activated repair and detoxifying systems, restricted access to or uptake
of drugs due to cell adhesion barriers, or enhanced drug efflux via broad
specificity, ATP-dependent pumps
(1). "Classical"
multidrug resistance is usually due to enhanced drug export by ATP-dependent
efflux pumps in the mdr, mrp, and related ABC transporter families.
However, it has become increasingly apparent that alterations in cell survival
and apoptotic signaling pathways are interconnected at many levels with
multidrug resistance mechanisms in cancer cells and that drug resistance in
patients may in some cases be overcome by therapeutic interventions that
induce downstream events in apoptotic cascades
(24).
Hyaluronan is a very large, linear glycosaminoglycan composed of repeating
disaccharides of glucuronic acid and N-acetylglucosamine. Although
hyaluronan is distributed ubiquitously in vertebrate tissues, both in the
embryo and in the adult, its organization with respect to cells is variable.
In adult tissues such as the vitreous, synovial fluid and the dermis, it
clearly plays an extracellular, structural role based on its unique
hydrodynamic properties. However, during dynamic cellular events such as
inflammation, wound repair, and tissue development, hyaluronan also interacts
with cells and influences their behavior in a variety of ways
(5,
6). In this context hyaluronan
binds cell surface receptors such as CD44 and RHAMM that transduce
intracellular signals, thus influencing cellular form and function directly
and instructively (6). In a
recent study, we found that perturbation of hyaluronan-cell interactions in
malignant cancer cells by treatment with hyaluronan oligosaccharides
(
12004000 Da) induces apoptosis under anchorage-independent
conditions and reduces tumor growth in vivo
(7). Hyaluronan oligomers
compete for endogenous polymeric hyaluronan, thus replacing high affinity,
multivalent and cooperative interactions with low affinity, low valency
receptor interactions (8,
9). We found that these
oligomers suppress the PI
3-kinase1/Akt cell
survival pathway, leading to proapoptotic events such as decreased
phosphorylation of BAD and FKHR, increased PTEN expression, and increased
caspase-3 activity (7). Current
evidence indicates that these effects are mainly due to disruption of
hyaluronan-CD44 interactions
(7), although it remains
possible that interactions with other extracellular, cell surface or
intracellular binding proteins are also involved. Nevertheless, since
multidrug resistance is often dependent on cell survival signaling pathways,
and since hyaluronan oligomers suppress at least part of these pathways, we
postulated that hyaluronan oligomers might reverse drug resistance.
 |
EXPERIMENTAL PROCEDURES
|
---|
Cell CultureMCF-7/Adr drug-resistant human mammary
carcinoma cells (obtained from Dr. K. Cowan, University of Nebraska) were
grown in culture for 24 h in 24-well plates in RPMI 1640 medium containing
Glutamax 1 plus 10% fetal bovine serum at 37o in 5% CO2.
Various concentrations of chemotherapeutic agents were then added and the
cells incubated for another 72 h, followed by a further 24 h in the presence
or absence of 100 µg/ml hyaluronan oligomers. The oligomers were a highly
purified, mixed population of three to eight repeating disaccharides in length
(7). Cells were harvested and
the number of viable cells counted in a Coulter Counter.
Recombinant Adenovirus InfectionMCF-7 cells were infected
overnight with a recombinant murine Has2 adenovirus, human emmprin
adenovirus, or control
-galactosidase adenovirus, which were
constructed and used as described previously
(10,
11). After changing the
medium, the cells were treated as described in the text and legends.
Assay of HyaluronanAfter infection with recombinant
adenoviruses as above, the cells were washed and then incubated for a further
48 h. Hyaluronan was measured in the medium and cell layer by an enzyme-linked
immunosorbent assay-like method
(12).
 |
RESULTS AND DISCUSSION
|
---|
We examined the effect of co-treatment with hyaluronan oligomers on drug
resistance in an established system, i.e. MCF-7/Adr human mammary
carcinoma cells that have been selected for resistance to doxorubicin
(13). First, we compared the
effect of hyaluronan oligomers on resistance to doxorubicin in the MCF-7/Adr
cells versus the relatively drug-sensitive, parental MCF-7 cell line.
We found that 100 µg/ml hyaluronan oligomers caused
55-fold
sensitization of the MCF-7/Adr cells to the drug, but had little effect on the
already sensitive MCF-7 cells (Fig. 1,
A and B;
Table I). We tested a range of
concentrations of the oligomers and found that, whereas concentrations up to
250 µg/ml have little or no effect on cell survival when used alone,
concentrations of 10 µg/ml or more have a highly significant effect on
doxorubicin resistance (data not shown). We then tested the effect of
hyaluronan oligomers on resistance of these cells to other drugs,
i.e. whether they affect multidrug resistance. We found that the
oligomers decreased resistance to taxol by
12-fold, to
1,3-bis(2-chloroethyl)-1-nitrosurea (BCNU) by
78-fold and to vincristine
by
10-fold (Table I).
Again, the oligomers had little effect on the parental MCF-7 cells
(Table I). In the case of taxol
there was only 23-fold difference in resistance between the two cell
types, yet only the MCF-7/Adr cells were affected significantly by the
oligomers. It is not yet clear why the MCF-7 cells were not sensitized by the
oligomers but this may be related to their endogenous levels of cell survival
pathway activity. We also tested the hyaluronan oligomers in a different cell
system, i.e. resistance of MDA-MB231 human mammary carcinoma cells to
the folate analog, methotrexate
(14). We found that they
decreased resistance in this system by 133-fold
(Fig. 1C;
Table I).

View larger version (19K):
[in this window]
[in a new window]
|
FIG. 1. Treatment with hyaluronan oligomers sensitizes drug-resistant carcinoma
cells to chemotherapeutic drugs. A, MCF-7/Adr drug-resistant
human mammary carcinoma cells were grown in the presence of various
concentrations of doxorubicin (Doxo) with or without 100 µg/ml
hyaluronan oligomers (o-HA), then cell numbers were measured.
B, MCF-7 human mammary carcinoma cells, which are much more sensitive
to drug treatment than the MCF-7/Adr cells
(13), were treated as
described in the legend to A. C, MDA-MB231 human mammary carcinoma
cells were treated with various concentrations of methotrexate in the presence
or absence of 100 µg/ml hyaluronan oligomers. The results in
AC are expressed as the means (±S.D.) of cell numbers
from three independent experiments performed in triplicate.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE I Effect of hyaluronan oligomers on IC50 values
MCF-7/Adr, MCF-7, or MDA-MB231 cells were treated with a range of drug
concentrations, with and without 100 µg/ml hyaluronan oligomers (o-HA), as
in Fig. 1A. All values
are expressed as µM.
|
|
If the hyaluronan oligomers sensitize multidrug-resistant cells by
perturbing hyaluronan interactions, then increased hyaluronan production might
be expected to cause increased resistance in drug-sensitive cells. Thus we
stimulated hyaluronan production in the relatively drug-sensitive MCF-7 cells
by infection with a recombinant adenovirus driving expression of HAS2, one of
the enzymes that synthesize hyaluronan
(15). In three separate
experiments, the Has2 adenovirus-infected cells were found to produce
2.54 times more hyaluronan than untreated cells or control cells
infected with recombinant
-galactosidase adenovirus and the increased
hyaluronan production induced a 1012-fold increase in resistance to
doxorubicin (Fig. 2A).
This increase in resistance was reversed by continuous treatment with
hyaluronan oligomers (Fig.
2B).
Recently we found that emmprin, a member of the Ig superfamily that is
enriched on the surface of most malignant cancer cells
(16) and that promotes tumor
progression (17), also
regulates hyaluronan production in tumor
cells.2 Thus we
infected MCF-7 cells with a recombinant emmprin adenovirus and found that
these cells were
10-fold more resistant to doxorubicin treatment than
controls (Fig. 2C).
The effect of emmprin was reversed by treatment with hyaluronan oligomers,
confirming that emmprin increases drug resistance via hyaluronan
(Fig. 2D).
In our previous work, we have found that hyaluronan oligomers suppress the
PI 3-kinase/Akt cell survival pathway. However these studies were performed in
different cells than those used here, i.e. HCT116 human colon
carcinoma and TA3/St mouse mammary carcinoma cells
(7). Thus we tested the effects
of the hyaluronan oligomers on this pathway in the MCF-7/Adr cells. As
expected, we found that hyaluronan oligomers suppress phosphorylation of Akt
and stimulate expression of PTEN in the presence of the various drugs,
i.e. doxorubicin, taxol, vincristine
(Fig. 3A), and BCNU
(not shown). PI 3-kinase activity was also inhibited but there were no effects
on total levels of Akt (not shown). These effects would be expected to lead to
decreased phosphorylation of BAD at serine residue 136 (BAD136), the site of
Akt-mediated phosphorylation. However, in MCF-7/Adr cells, as opposed to the
cells used previously, we found very little phosphorylation of BAD136 in the
presence or absence of the drugs or oligomers (not shown). Thus we examined
the MAP kinase pathway, which also leads to BAD phosphorylation, in this case
at serine 112 by Erk
(1820).
We found strong phosphorylation of BAD112 in the MCF-7/Adr cells treated with
the various drugs, implying that the MAP kinase pathway is more involved in
phosphorylation of BAD than the PI 3-kinase pathway in these cells. We also
found that BAD112 phosphorylation is inhibited by the hyaluronan oligomers
(Fig. 3, A and
B). In addition we found that the oligomers inhibit
phosphorylation, but not total levels, of upstream components of this pathway,
i.e. Erk (Fig. 3, A and
B) and Raf-1 (not shown), in the presence of the various
drugs. Since these experiments were done under anchorage-dependent culture
conditions, we would expect the MAP kinase pathway to be activated by FAK
(21,
22). Therefore we examined FAK
phosphorylation in drug-treated MCF-7/Adr cells in the presence and absence of
hyaluronan oligomers and found that the oligomers inhibit phosphorylation of
FAK (Fig. 3A).
Inhibition of p-Erk, p-BAD112, and p-FAK levels in these experiments varied
from 5090% depending on drug and oligomer dosage. Experiments were also
performed with 501000 nM BCNU, and similar inhibition was
observed (not shown).

View larger version (38K):
[in this window]
[in a new window]
|
FIG. 3. Hyaluronan regulates cell survival pathways. A, MCF-7/Adr
cells, treated in the presence of various drugs with and without 100 µg/ml
hyaluronan oligomers (o-HA) as in
Fig. 1A and
Table I, were processed for
Western blot analysis of phosphorylated Akt (p-Akt), p-Erk, p-BAD112,
p-FAK, and PTEN. Similar results were obtained for PTEN with taxol and
vincristine (not shown). Doxorubicin (Doxo): lane 1, 20
nM; lane 2, 20 nM + hyaluronan oligomers;
lane 3, 100 nM; lane 4, 100 nM +
hyaluronan oligomers. Taxol: lane 1, 20 nM; lane
2, 20 nM + hyaluronan oligomers; lane 3, 100
nM; lane 4 100 nM + hyaluronan oligomers.
Vincristine: lane 1, 20 nM; lane 2, 20
nM + hyaluronan oligomers; lane 3, 100 nM;
lane 4, 100 nM + hyaluronan oligomers. Similar results
were obtained with p-Raf-1 but no changes in total levels of Akt or Raf-1 were
observed (not shown). B, MCF-7/Adr cells were treated with and
without 50 nM doxorubicin in the presence of 0100 µg/ml
hyaluronan oligomers and processed for Western blotting for p-Erk and
p-BAD112. Lane 1, no additions; lane 2, 50 nM
doxorubicin only; lanes 35, 50 nM doxorubicin + 1
µg/ml hyaluronan oligomers (lane 3), 10 µg/ml hyaluronan
oligomers (lane 4)or100 µg/ml hyaluronan oligomers (lane
5). C, MCF-7 cells infected with recombinant Has2 or
-galactosidase adenovirus were processed for Western blot analysis of
p-Akt, p-Erk, p-BAD112, and p-FAK. Lanes 13,
-galactosidase; lanes 46, Has2. Lanes 1 and 4,
no addition; lanes 2 and 5, 20 nM doxorubicin;
lanes 3 and 6, 100 nM doxorubicin. Each of the
experiments in AC was repeated at least three times and
similar results were obtained.
|
|
Since increased hyaluronan production causes enhanced drug resistance in
MCF-7 cells, we also determined whether these pathways were stimulated in
recombinant Has2 adenovirus-infected cells. As expected, we found
that phosphorylation of Akt, Erk, BAD112, and FAK was increased in
drug-treated, Has2 adenovirus-infected cells compared with controls
(Fig. 3C), whereas
PTEN expression was decreased (not shown). Similar results were obtained with
emmprin adenovirus-infected cells.
The experiments described above indicate that endogenous hyaluronan-tumor
cell interactions are a crucial component of the regulation of multidrug
resistance in cancer cells and that the most likely mechanism whereby
hyaluronan acts is by stimulating the PI 3-kinase and MAP kinase cell survival
pathways, leading to various anti-apoptotic consequences such as
phosphorylation of BAD. Active, non-phosphorylated BAD interacts with
prosurvival Bcl-2 family members and induces apoptosis
(23). BAD is inactivated by
phosphorylation at serine 136 by Akt or at serine 112 by Erk, either of which
leads to anti-apoptotic consequences that can result in increased drug
resistance in tumor cells
(1820).
In the MCF-7/Adr human mammary carcinoma cells used here, regulation of BAD
phosphorylation is mediated mainly by Erk. However, in HCT116 human colon
carcinoma and TA3/St mouse mammary carcinoma cells, phosphorylation of BAD by
Akt is prominent (7). In either
case, treatment of the cells with hyaluronan oligomers is inhibitory.
The idea that hyaluronan-cell interactions may be related to drug
resistance is also supported by past data showing that hyaluronidase enhances
the action of various chemotherapeutic agents, especially when used locally
(24). Of particular interest
is the observation that dispersion of multicellular spheroids of EMT-6 mammary
tumor cells with hyaluronidase reverses MDR1-based multidrug resistance
(25,
26). The mechanistic effect of
hyaluronidase in these systems is not understood but has been explained in
terms of decreased cell adhesion
(25) or increased drug
penetration (24,
27), rather than
hyaluronan-specific effects on cell survival signaling. In earlier work we
showed that calcium-independent aggregation of transformed cells is due to
hyaluronan-mediated, multivalent cross-bridging of receptors on adjacent cells
(5,
28). Thus it is probable that
hyaluronan-induced promotion of the PI 3-kinase/Akt or MAP kinase cell
survival pathways is responsible for enhanced drug resistance in these
spheroids, in addition to or instead of restricted drug access. Recent work
shows that enhanced integrin signaling can also induce drug resistance
(29). We show here that
perturbation of endogenous hyaluronan-induced signaling inhibits FAK
phosphorylation, a central event in integrin signaling
(21,
22). Also, we have shown that
hyaluronan signaling is critical for anchorage-independent growth
(7), a phenomenon that has
previously been related to altered integrin signaling
(21,
22,
30). These observations
suggest that hyaluronan-mediated signaling and integrin signaling play
overlapping or interacting roles in these events.
Our results not only document a role for hyaluronan in multidrug
resistance, they also indicate that perturbation of hyaluronan interactions
sensitizes resistant cells. Thus, such perturbations may provide a dual
therapeutic role, since they have an intrinsic effect on tumor growth and
metastasis (31,
32), as well as sensitizing
cancer cells to chemotherapeutic agents as shown herein.
 |
FOOTNOTES
|
---|
* This work was supported by National Institutes of Health Grants CA73839,
CA82867, and CA79866 (to B. P. T.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed: Cell Biology and Anatomy, Medical
University of South Carolina, 173 Ashley Ave., Charleston, SC 29425. Tel.:
843-792-3521; Fax: 843-792-0664; E-mail:
toolebp{at}musc.edu.
1 The abbreviations used are: PI 3-kinase, phosphoinositide 3-kinase; BAD136,
serine residue 136 of BAD; BAD112, serine residue 112 of BAD; FAK, focal
adhesion kinase; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosurea; MAP,
mitogen-activated protein. 
2 E. Marieb, A. Zoltan-Jones, S. Misra, S. Ghatak, R. Li, and B. P. Toole,
manuscript in preparation. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. K. Cowan, University of Nebraska, for donating the MCF-7/Adr
and MCF-7 parental cell lines; Drs. Lei Huang, Rongsong Li, and Ken Walsh for
the adenoviral constructs; Dr. Leslie Gordon and Ingrid Harten for help with
the hyaluronan assays; and Dr. J. Castellot for use of the Coulter
Counter.
 |
REFERENCES
|
---|
- Gottesman, M. M., Fojo, T., and Bates, S. E. (2002)
Nat. Rev. Cancer 2,
4858[CrossRef][Medline]
[Order article via Infotrieve]
- Lowe, S. W., and Lin, A. W. (2000)
Carcinogenesis 21,
485495[Abstract/Free Full Text]
- Makin, G., and Dive, C. (2001) Trends Cell
Biol. 11,
S22S26[CrossRef][Medline]
[Order article via Infotrieve]
- O'Gorman, D. M., and Cotter, T. G. (2001)
Leukemia (Baltimore) 15,
2134[CrossRef][Medline]
[Order article via Infotrieve]
- Toole, B. P. (2001) Semin. Cell Dev.
Biol. 12,
7987[CrossRef][Medline]
[Order article via Infotrieve]
- Turley, E. A., Noble, P. W., and Bourguignon, L. Y.
(2002) J. Biol. Chem.
277,
45894592[Free Full Text]
- Ghatak, S., Misra, S., and Toole, B. P. (2002)
J. Biol. Chem. 277,
3801338020[Abstract/Free Full Text]
- Underhill, C. B., Chi-Rosso, G., and Toole, B. P.
(1983) J. Biol. Chem.
258,
80868091[Abstract/Free Full Text]
- Lesley, J., Hascall, V. C., Tammi, M., and Hyman, R.
(2000) J. Biol. Chem.
275,
2696726975[Abstract/Free Full Text]
- Li, R., Huang, L., Guo, H., and Toole, B. P. (2001)
J. Cell. Physiol. 186,
371379[CrossRef][Medline]
[Order article via Infotrieve]
- Ward, J., Huang, L., Guo, H., Ghatak, S., and Toole, B. P.
(2003) Am. J. Pathol.
162,
14031409[Abstract/Free Full Text]
- Kongtawelert, P., and Ghosh, P. (1990)
Anal. Biochem. 185,
313318[Medline]
[Order article via Infotrieve]
- Fairchild, C. R., Ivy, S. P., Kao-Shan, C. S., Whang-Peng, J.,
Rosen, N., Israel, M. A., Melera, P. W., Cowan, K. H., and Goldsmith, M. E.
(1987) Cancer Res.
47,
51415148[Abstract]
- Worm, J., Kirkin, A. F., Dzhandzhugazyan, K. N., and Guldberg, P.
(2001) J. Biol. Chem.
276,
3999040000[Abstract/Free Full Text]
- Weigel, P. H., Hascall, V. C., and Tammi, M. (1997)
J. Biol. Chem. 272,
1399714000[Free Full Text]
- Biswas, C., Zhang, Y., DeCastro, R., Guo, H., Nakamura, T.,
Kataoka, H., and Nabeshima, K. (1995) Cancer
Res. 55,
434439[Abstract]
- Zucker, S., Hymowitz, M., Rollo, E. E., Mann, R., Conner, C. E.,
Cao, J., Foda, H. D., Tompkins, D. C., and Toole, B. P. (2001)
Am. J. Pathol. 158,
19211928[Abstract/Free Full Text]
- Bonni, A., Brunet, A., West, A. E., Datta, S. R., Takasu, M. A.,
and Greenberg, M. E. (1999) Science
286,
13581362[Abstract/Free Full Text]
- Mabuchi, S., Ohmichi, M., Kimura, A., Hisamoto, K., Hayakawa, J.,
Nishio, Y., Adachi, K., Takahashi, K., Arimoto-Ishida, E., Nakatsuji, Y.,
Tasaka, K., and Murata, Y. (2002) J. Biol.
Chem. 277,
3349033500[Abstract/Free Full Text]
- Masters, S. C., Yang, H., Datta, S. R., Greenberg, M. E., and Fu,
H. (2001) Mol. Pharmacol.
60,
13251331[Abstract/Free Full Text]
- Tamura, M., Gu, J., Tran, H., and Yamada, K. M. (1999)
J. Natl. Cancer Inst.
91,
18201828[Abstract/Free Full Text]
- Almeida, E. A., Ilic, D., Han, Q., Hauck, C. R., Jin, F.,
Kawakatsu, H., Schlaepfer, D. D., and Damsky, C. H. (2000)
J. Cell Biol. 149,
741754[Abstract/Free Full Text]
- Datta, S. R., Brunet, A., and Greenberg, M. E. (1999)
Genes Dev. 13,
29052927[Free Full Text]
- Baumgartner, G., Gomar-Hoss, C., Sakr, L., Ulsperger, E., and
Wogritsch, C. (1998) Cancer Lett.
131,
8599[CrossRef][Medline]
[Order article via Infotrieve]
- St Croix, B., Rak, J. W., Kapitain, S., Sheehan, C., Graham, C. H.,
and Kerbel, R. S. (1996) J. Natl. Cancer
Inst. 88,
12851296[Abstract/Free Full Text]
- St Croix, B., Man, S., and Kerbel, R. S. (1998)
Cancer Lett. 131,
3544[CrossRef][Medline]
[Order article via Infotrieve]
- Desoize, B., and Jardillier, J. (2000)
Crit. Rev. Oncol. Hematol.
36,
193207[Medline]
[Order article via Infotrieve]
- Underhill, C. B., and Toole, B. P. (1981)
Exp. Cell Res. 131,
419423[Medline]
[Order article via Infotrieve]
- Damiano, J. S., Hazlehurst, L. A., and Dalton, W. S.
(2001) Leukemia
15,
12321239[CrossRef][Medline]
[Order article via Infotrieve]
- Frisch, S. M., and Screaton, R. A. (2001)
Curr. Opin. Cell Biol.
13,
555562[CrossRef][Medline]
[Order article via Infotrieve]
- Toole, B. P. (2002)
Glycobiology 12,
37R42R[Abstract/Free Full Text]
- Toole, B. P., and Hascall, V. C. (2002) Am.
J. Pathol. 161,
745747[Free Full Text]