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Table 1 Relationship between ALDOC expression and clinicopathologic characteristics
ALDOC expression
Positive
Age
≤65 (n=63)
Negative
p-value
36 (57%)
27
30
42
39 (47%)
27
44
25
0.5767
21 (54%)
18
0.7068
45
51
59 (48%)
63
0.4626
13 (33%)
27
0.0127
53
42
26 (42%)
40
36
33
0.1357
M0 (n=107)
M1 (n=28)
Lymphatic invasion
46 (43%)
20
61
0.0067
ly0 (n=59)
ly1/2/3 (n=76)
Vascular invasion
25 (42%)
41
34
35
0.1814
v0 (n=66)
v1/2/3 (n=69)
31 (47%)
35
35
34
0.6626
24 (41%)
42
34
35
0.1291
>65 (n=72)
0.0722
Sex
Male (n=83)
Female (n=52)
Tumor Location
Right colon (n=39)
Left colon (n=96)
Differentiation
Differentiated (n=122)
Un-differentiated (n=13)
T grade
T1/T2 (n=40)
T3/T4 (n=95)
N grade
N0 (n=62)
N1/N2/N3 (n=73)
M grade
Stage
Stage I/II (n=58)
Stage III/IV (n=77)
p-values were calculated with Chi-square test.
Maruyama R et al. 23
Table 2 Univariate and multivariate Cox regression analysis of survival
Univariate analysis
HR
Multivariate analysis
95 % CI
p-value
(0.586-2.170)
0.7182
(0.293-1.210)
0.1403
(0.325-1.267)
0.212
(0.332-3.541)
0.8948
HR
95 % CI
p-value
(0.336-7.728)
0.5515
(0.132-1.004)
0.2695
(2.714-13.916)
< 0.001
(0.843-4.363)
0.1206
(1.129-43.160)
0.0436
(1.010-4.476)
0.0400
Age
≤65
1 (Ref.)
>65
1.128
Sex
Male
Female
1 (Ref.)
0.595
Tumor Location
Right colon
1 (Ref.)
Left colon
0.641
Differentiation
Differentiated
Un-differentiated
1 (Ref.)
1.084
T grade
T1/T2
1 (Ref.)
T3/T4
7.865
1(Ref.)
(1.888-32.765)
0.0001
1.61
N grade
N0
N1/N2/N3
1 (Ref.)
5.227
1 (Ref.)
(2.172-12.577)
<0.001
0.482
M grade
M0
1 (Ref.)
M1
14.001
1 (Ref.)
(6.825-28.720)
< 0.001
6.146
Lymphatic invasion
ly0
ly1/2/3
1 (Ref.)
3.05
1 (Ref.)
(1.432-6.493)
0.0019
(0.805-3.736)
0.0697
1.918
Vascular invasion
v0
v1/2/3
1 (Ref.)
1.867
Stage
Stage I/II
1 (Ref.)
Stage III/IV
10.984
1 (Ref.)
(3.363-35.881)
< 0.001
6.98
ALDOC expression
Negative
1 (Ref.)
Positive
3.104
1 (Ref.)
(1.521-6.337)
HR, hazard ratio; CI, confidence interval.
0.0011
2.126
Maruyama R et al. 24
Table 3 Correlation between ALDOC expression and CD44
ALDOC expression
Positive
Negative
CD44
Positive
Negative
p-value
0.0091
41(59%)
25(38%)
p-values were calculated with Chi-square test.
29
40
Maruyama R et al. 25
Figure legends
Figure 1. Effects of ALDOC inhibition on cell growth and spheroid formation in colorectal
cancer (CRC) cell lines. (a) qRT-PCR analysis of ALDOC in DLD-1 and WiDr. Fold-change
indicates the ratio of ALDOC mRNA expression in spheroid body-forming cells to that in
attached parental cells. (b) Western blot analysis of ALDOC in five CRC cell lines. (c) Western
blot analysis of ALDOC in RKO and LoVo cells transfected with the ALDOC-specific siRNA
and negative control siRNA. (d) Cell growth was assessed by MTT assays at days 1, 2, and 4
after transfection and seeding in 96-well plates. Data are presented as means ± standard
deviations of 3 independent experiments. * p < 0.05. (e) The numbers and size of spheroids
formed by RKO and LoVo cells transfected with ALDOC-specific or negative control siRNAs.
Data are presented as means ± standard deviations of 3 independent experiments. * p < 0.05.
Figure 2. Effects of ALDOC inhibition on migration of and lactate production in CRC cells.
(a) Wound healing assays for RKO cells transfected with the ALDOC siRNA or negative
control siRNA. The migration of cells was captured at 0 h and 24 h after scratching. (b) Wound
healing assays for LoVo cells transfected with the ALDOC siRNA or negative control siRNA.
The migration of cells was captured at 0 h and 48 h after scratching. (c) Images of invasion
assay in RKO cells transfected with the ALDOC siRNA or negative control siRNA at 48 h and
Maruyama R et al. 26
quantification of the average number of invaded cells. (d) Images of invasion assay in LoVo
cells transfected with the ALDOC siRNA or negative control siRNA at 48 h and quantification
of the average number of invaded cells. (e) RKO cells and (f) LoVo cells transfected with the
ALDOC siRNA and negative control siRNA were cultured for 1 day, and the lactate
concentration in the culture medium was analyzed. Data are presented as means ± standard
deviations of 3 independent experiments. * p < 0.05.
Figure 3. Immunohistochemical analysis of ALDOC in colorectal cancer (CRC) tissue samples.
(a) Immunohistochemical analysis of ALDOC in CRC. Left panel, original magnification, ×40.
Right panel, a high-magnification image of the field designated by the box in the left panel;
original magnification, ×400. (b) Kaplan-Meier plots of the survival of CRC patients. (c)
Immunohistochemical analysis of ALDOC and CD44. Original magnification, ×400.
Maruyama R et al. 27
Figure 1
Maruyama R et al. 28
Figure 2
Maruyama R et al. 29
Figure 3
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