Download Hyaluronan Oligosaccharides Inhibit Tumorigenicity of Breast

Hyaluronan Oligosaccharides Inhibit Tumorigenicity of Breast Cancer Cells
in Vitro and Metastatic Lesions of Bone in Vivo
1
Urakawa, H; +1Nishida, Y; 1Wasa, J; 1Arai, E; 2Suzuki, Y; 3Hosono, K; 1Kozawa, E; 1Futamura, N; 1Ishiguro, N
+1Orthopaedics, Nagoya University, Nagoya, Japan, 2 Orthopaedics, Kariya Toyota General Hospital, Kariya, Japan,
3
Orthopaedics, Nagoya Memorial Hospital, Nagoya, Japan
Senior author: [email protected]
HA10 and HMWHA.HABP staining: The HA in MDA-MB-231 cells
INTRODUCTION:
In malignant tumors, hyaluronan (HA) is known to be abundantly
was located on cell surface and in cytoplasms. Stainable HA was
accumulated in tumor parenchyma and surrounding stromal cells.
decreased after 72h treatment with HA10 and HMWHA (Fig. 3 A-D).
Previous studies demonstrated that HA oligosaccharides (oligos)
Real time RT-PCR: Treatments with HA4 and 10 significantly
suppress the tumorigenicity of various malignant tumors via disruption
downregulated expression level of HAS2 mRNA at 5 days. Those of
of receptor-HA interaction. However, few studies have focused on breast
HAS3 and CD44 mRNA were not significantly downregulated. In vivo
cancer and its bone metastasis. We investigated the critical size of HA
experiments: HA10 inhibited the progression of osteolytic lesion by the
oligos to induce antitumor effects in highly invasive breast cancer cell
tumor on soft X-ray (Fig.4A), and also suppressed HA accumulation in
line, MDA-MB-231 cells. In addition, the effect of HA oligos on bone
the tumor (Fig. 4B, C).
metastasis of breast cancer were analyzed in vivo using murine model.
MATERIAL AND METHODS:
Cell culture: MDA-MB-231 cells (human breast cancer cell line) was
maintained at 37℃ in an atmosphere with 5% CO2 with DMEM,
supplemented with 10% fetal bovine serum, penicillin, and streptomycin.
B
A
C
Retention of Exogenous Fluoresceinamine Labeled HA
Figure 1. Exogenous Fl-HA accumulation in MDA-MB-231 breast
oligosaccharides: MDA-MB-231 cells were pretreated with an anticancer cells. (A) 0μg/ml, (B) 5μg/ml, and (C) 10μg/ml Hermes-1.
CD44 neutralizing antibody (Hermes-1) for 2 hours at r.t. and cultured
on chamber slides at a density of 5×104 cells / well with Fl-HA oligos
A
B
(Mw: 20 to 30kd) for 24 hours and then observed using a fluorescent
microscope. Proliferation assay: MDA-MB-231 cell line was seeded in
96-well plate at 1×104 cells/well. Cell growth in medium with or
without 250 μg/ml various size of HA oligos or high molecular weight
HA (HMWHA) was measured by MTT [3-(4,5-dimethylthiazol)-2,5diphenyltetrazolium bromide] assay (Boeringer Mannheim, Germany) at
24, 48, and 72h. Motility and matrigel invasion assays: Chemotactic
motility was investigated using 24-well culture chambers containing
inserts with 12μm pores. Cells were added to the upper chamber with
Figure 2. (A) MTT assay. Data are expressed as mean +/- S.D. *
or without 250 μg/ml HA4, HA10, and HMWHA. Invasion was
p<0.05 and **p<0.001 compared with control. (B) Effects of HA oligos
assayed in the same chambers that also contained a Matrigel layered on
or HMWHA on Akt phosphorylation in MDA-MB-231 cells pretreated
a 12μm pore membrane. Migration and invading cells on the lower
with or without 10 µg/ml Hermes-1.
surface of the membrane were counted under the light microscopy at 24h
after incubation. Western blot analysis : MDA-MB-231 cells cells were
pretreated with or without 10µg/ml Hermes-1 for 1 hour at r.t, and then
protein was collected at 2h after treatments with or without 250 μg/ml
HA4, HA10, or HMWHA. Target proteins were visualized with specific
antibodies to p-Akt, Akt and beta-actin individually. Densitometric
A
B
C
D
analysis of the immunoreactive areas was performed and the results are
Figure 3. HABP staining of MDA-MB-231 breast cancer cells. (A)
expressed as the index (p-Akt / beta-actin). HABP staining: HA
contorol, (B) HA4, (C) HA10, and (D) HMWHA.
accumulation in cultured cells or bone metastasis in vivo model was
A
C
B
visualized using Hyaluronic Acid Binding Protein (HABP, Seikagaku,
Japan). Real time RT-PCR: Levels of mRNA expression for CD44,
HAS2, and HAS3 by MDA-MB-231 cells with or without 250 μg/ml
HA4, HA10, and HMWHA for 5days was assessed by real time RTPCR method, normalized with mRNA level of GAPDH expression. In
vivo experiments: MDA-MB-231 cells were implanted into tibia of 4week old BALB/C mice. After confirmation of osteolytic lesion on XFigure4. (A) Osteolytic lesion of tibia after treatment with or without
ray, direct injection of 20µl serum free DMEM with or without
HA10 (n=8 in each group). * p<0.01. HABP staining of tumor in vivo
250µg/ml HA10 was performed into the tumor in tibia every 2 days for
bone metastasis model after direct injection of control medium (B) or
14 days. After 14 days of consecutive injection, the mice were subjected
HA10 (C).
to soft X-ray evaluation, and histologic examination. Statistical
DISCUSSION:
analysis: Bonferroni-Dunn post-hoc test and unpaired Student’s t test
Manipulation of HA expression was previously described to inhibit
were used to assess differences between groups. P-values of <0.05 were
malignant properties in breast cancer cell lines [1]. In our study, HA10
considered as statically significant.
downregulated HA expression in vitro and in vivo bone metastasis
RESULTS:
model in human breast cancer cells. HA-CD44 binding has been also
Retention of Exogenous Fl- HA oligos: The retention of HA oligos in
reported to influence the activity of PI3K/Akt pathway and be perturbed
cytoplasm was specifically blocked in a dose-dependent manner using
by treatment with HA oligos [2, 3]. These reports and our data suggest
neutralizing anti-CD44 monoclonal antibody, Hermes-1 (Fig. 1A-C).
that decreased HA expression and HA-CD44 interruption by HA oligos
Proliferation assay: The differences in cell viability between control
reduced tumorigenicity of breast cancer in vitro and particularly in vivo
and HA6,8,10,12, HMWHA were statistically significant at 48h and/or
bone metastasis model. HA-CD44 interruption by HA oligos can be one
72h after incubation (p<0.05) (Fig. 2A). Motility and matrigel invasion
of the therapeutic candidates for bone metastasis of breast cancer.
assays: Incubation with HA10 and HMWHA suppressed both motility
REFFERENCES:
and invasiveness significantly compared with those of control. Western
1.Udabage L et al. Cancer Research. 2005;65: 6139-50.
blot analysis: Treatments with HA10 and HMWHA decreased 24 and
2. Russo RIC et al. International Journal of Cancer. 2008;122: 1012-18.
17% of the p-Akt level, respectively (Fig. 2 B). Pretreatment with anti
3. Ghatak S et al. Journal of Biological Chemistry. 2002;277: 38013-20.
human CD44 monoclonal antibody partially abolished the effect of
Paper No. 435 • ORS 2011 Annual Meeting