Dig Dis Sci (2013) 58:2713–2720 DOI 10.1007/s10620-013-2692-z
ORIGINAL ARTICLE
Increased a-Tubulin1b Expression Indicates Poor Prognosis and Resistance to Chemotherapy in Hepatocellular Carcinoma Cuihua Lu • Jing Zhang • Song He • Chunhua Wan Aidong Shan • Yingying Wang • Litao Yu • Guoliang Liu • Ken Chen • Jing Shi • Yixin Zhang • Runzhou Ni
•
Received: 5 February 2013 / Accepted: 13 April 2013 / Published online: 27 April 2013 Ó Springer Science+Business Media New York 2013
Abstract Background Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide. It is important to understand molecular mechanisms of HCC progression and to develop clinically useful biomarkers for the disease. Aim We aimed to investigate the possible involvement of a-tubulin1b (TUBA1B) in HCC pathology. Methods Tissue specimens were obtained from 114 HCC patients during hepatectomy. Immunohistochemistry and western blot analysis were used to detect TUBA1B expression in HCC tissues and cell lines. TUBA1B was knocked down in HCC cells by siRNA transfection. CCK-8 assay and flow cytometry were applied to determine cell proliferation and cell cycle progression, respectively. The efficacy of paclitaxel chemotherapy was evaluated by plate colony formation assay. Results TUBA1B was higher expressed in HCC tumor tissues than in adjacent nontumor tissues. TUBA1B and Ki-67 expressions were positively related to each other, and both their expressions were significantly associated with histological grade of HCC patients. Univariate and multivariate survival analyses revealed that TUBA1B was a significant predictor for overall survival of HCC patients.
Cuihua Lu and Jing Zhang contributed equally to this work. C. Lu J. Zhang C. Wan A. Shan Y. Wang L. Yu G. Liu K. Chen J. Shi R. Ni (&) Department of Gastroenterology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province, People’s Republic of China e-mail:
[email protected] S. He Y. Zhang Department of Pathology, Nantong University Cancer Hospital, Nantong, Jiangsu Province, People’s Republic of China
TUBA1B expression was increased in HCC cells during the G1- to S-phase transition. TUBA1B knockout in HCC cells inhibited cell proliferation, and attenuated resistance to paclitaxel. Conclusions Our results indicated that TUBA1B expression was upregulated in HCC tumor tissues and proliferating HCC cells, and an increased TUBA1B expression was associated with poor overall survival and resistance to paclitaxel of HCC patients. Keywords Hepatocellular carcinoma a-Tubulin1b (TUBA1B) Ki-67 Prognosis Chemotherapy
Introduction Hepatocellular carcinoma (HCC) is one of the most common human cancers worldwide with steadily increasing incidence in recent years [1]. HCC ranks as the third cause of mortality among deaths from cancer. Although surgical resection and liver transplantation are used for treating HCC, the overall prognosis of HCC patients is very grim due to many risk factors, including late diagnosis, extensive tumor invasion, high recurrence, frequent extrahepatic metastasis, and resistance to chemotherapy [2–4]. Therefore, significant research efforts have been devoted to understanding the molecular mechanism of HCC progression and searching for clinically useful biomarkers to predict the prognosis of HCC patients. Tubulin is a major cytoskeleton component with 5 distinct forms designated a-, b-, c-, d-, and e-tubulin. a- and b-tubulin heterodimers form the microtubules, which are involved in cell adhesion, movement, replication, and division [5]. Very importantly, microtubules are in a dynamic process of polymerization and depolymerization
123
2714
during cell replication and division, and, if the dynamic process of microtubules is broken, cell proliferation and differentiation are affected [6]. The mitotic spindle, polymerized by microtubules in the process of cell division, pulls chromosomes into two daughter cells during cell mitosis [7]. Since microtubules play important roles in the regulation of the mitotic apparatus, microtubule disruption causes a cell cycle arrest in G2/M phase and results in the formation of abnormal mitotic spindles, thus triggering apoptosis signals [8]. Hence, highly dynamic mitotic spindle microtubules are among the most successful targets for anticancer therapy. The most active anticancer drugs, such as taxanes and vinca alkaloids, perturb the dynamic equilibrium of microtubule polymerization and depolymerization, and thus exert their functions [9–11]. Unfortunately, the efficacy of these anticancer drugs is often limited by multidrug resistance in tumors [12–14]. Recently, several members of the tubulin family have proven to be involved in the progression of different tumors, and representative examples are listed here: (1) class III b-tubulin (TUBB3) is highly expressed in ovarian carcinoma, non-small-cell lung cancer, thymic epithelia tumor, breast cancer and other tumors [15–18], and TUBB3 expression is significantly higher in tumor stages III–IV than in tumor stages I–II; (2) delocalization of c-tubulin induced by an increased solubility in breast cancer cells yields a greater resistance to microtubule depolymerization with colchicine [19]; (3) tubulin, alpha 3c (TUBA3C) expression reduces the sensitivity to paclitaxel chemotherapy in ovarian cancer patients [20]; and (4) ka1 tubulin expression is slightly increased in papillary carcinomas but greatly increased in anaplastic carcinomas, and so microtubuletargeted chemotherapeutic drugs may be used for treating anaplastic carcinomas [21]. Driven by these findings, we hypothesized that there may exist a tubulin isoform, whose expression was associated with the prognostic value and chemotherapy efficacy in HCC. To test our hypothesis, atubulin1b (TUBA1B) was chosen as the subject of this study. We determined TUBA1B expression in HCC tumor tissues and cell lines, and evaluated the prognostic significance of TUBA1B expression in HCC. After small interfering RNA (siRNA) was used to knockdown TUBA1B expression in HCC cells, we further observed the effects of decreased TUBA1B expression on cell proliferation and chemotherapeutic sensitivity of HCC cells.
Dig Dis Sci (2013) 58:2713–2720
curative surgery at the Affiliated Hospital of Nantong University. After surgical removal, a portion of paired tissue samples were snap-frozen in liquid nitrogen and then maintained at -80 °C until use for protein extraction, and another portion of paired tissue samples were immediately fixed in formalin and embedded in wax for immunohistochemistry. The inclusion and exclusion criteria for all patients in this study were set as follows: (1) diagnosis was confirmed by experienced pathologists through histological examination of H&E-stained biopsy sections; (2) there were no anticancer treatments before surgery; (3) curative resection was defined as the macroscopically complete removal of the tumor and a histologically proven tumor-free margins; and (4) clinicopathologic and follow-up data were completely available. Informed consent was obtained from each patient, and study protocols were approved by the Institutional Review Board (IRB) of Affiliated Hospital of Nantong University. The patients included 91 males and 23 females with a mean age of 49.3 years, range 21–75 years. The main clinicopathological variables of subjects were recorded. Histological grades were classified to well(grade I, n = 6), moderately- (grade II, n = 54), and poorly- (grade III, n = 54) differentiated tumors. The follow-up time was 1 to 96 months. Immunohistochemistry
Methods
Immunohistochemistry was performed as described previously [22]. In brief, paired tissue sections were dewaxed, washed, and blocked. Afterwards, sections were incubated for 1 h at room temperature with primary antibodies: antiTUBA1B (1:1,000) or anti-Ki-67 (1:100, both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish peroxidase (HRP)-conjugated species-specific second antibodies. The slides were then mounted for observation under a fluorescence microscope. All immunostained sections were independently examined by two pathologists in a blinded manner without knowledge of the clinicopathological variables of patients. At least 10 highpower fields in each specimen were randomly selected, and the nuclear or cytoplasma staining was observed under a high magnification. For determining TUBA1B expression, the intensity of immunostaining was assessed as strong (3), moderate (2), weak (1), or negative (0). For determining Ki-67 expression, more than 500 cells were counted to determine the labeling index, which was expressed as the percentage of the immunostained cells over total cells.
Patients and Tissue Samples
Cell Culture and Cell Transfection
The paired samples of tumor and adjacent nontumor tissues were obtained from 114 HCC patients who had underwent
Two normal hepatocyte cell lines (HL-7702 and chang) and 5 HCC cell lines (SMMC-7721, HepG2, BEL-7404,
123
Dig Dis Sci (2013) 58:2713–2720
Hep3B and HuH7) were provided by the Institute of Cell Biology of the Chinese Academy of Sciences. The cells were respectively cultured in RPMI 1640 and Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin, and 100 lg/mL streptomycin (all mediums from Invitrogen, Carlsbad, CA, USA) in 5 % CO2 at 37 °C. Two human TUBA1B siRNA expression vectors and pSilencer 4.1-CMV neo-TUBA1B siRNA were constructed, respectively. The siRNA targeting the nucleotide residues 50 -AGTTCTCCATTTACCCAGCA-30 and 50 -AAAACCAAGC GCAGCATCCA-30 , named siRNA#1 and #2, respectively. HuH7 cells were seeded the day before transfection using DMEM with 10 % FBS but without antibiotics. Transfection was performed using lipofectamine 2,000 transfection reagent (Invitrogen) according to the manufacture’s protocol. The cells were incubated with the pSilencer vectors and lipofectamine plus reagent complexes for 4–6 h at 37 °C, and FBS was added to DMEM at a final concentration of 10 %. The cells were used for subsequent experiments at 48 h after transfection. Western Blot Analysis Experimental protocols were performed as described previously [22]. In brief, protein extracts from tissues or cells were subjected to protein quantification using a BCA kit (Pierce, Rockford, IL, USA). Then the protein samples were resolved by SDS-PAGE and transferred to a PVDF membrane, which was incubated with primary antibodies: anti-TUBA1B (1:500) or anti-Ki-67 (1:100, both from Santa Cruz Biotechnology) for 2 h at room temperature, followed by reaction with HRP-conjugated secondary antibodies. The detection of specific signals was performed using an ECL method. The resulting bands on the autoradiograph were analyzed by densitometry, and normalized against b-actin or GAPDH (loading control).
2715
by flow cytometry using a Becton–Dickinson FACScan and Cell Quest acquisition and analysis programs. Gating was set to exclude cell debris, cell doublets, and cell clumps. Measurement of Drug Resistance To determine the dose responses to chemotherapeutic drugs, an equal number of cells (*100–200) were plated onto 6 wells of a 24-well dish containing increasing concentrations of paclitaxel (0–400 ng/mL), which were selected by referring to previous study results [14]. After incubation for 7 days at 37 °C, the medium was removed, and the cells were stained with 0.5 % crystal violet as described previously [23]. The dish was rinsed to remove excess stain, and the cells were observed and photographed with a Coolpix s2500 digital camera (Nikon, Melville, NY, USA). Statistical Analysis Statistical analysis was performed using the Stat View 5.0 software package. The association between TUBA1B expression and clinicopathological variables was analyzed using the Pearson v2 test. The relationship between TUBA1B and Ki-67 expressions was studied using the Spearman rank correlation test because the data were not normally distributed. Survival analysis was performed using the Kaplan–Meier method, and the difference in survival rates was assessed by the generalized log-rank test [24]. Multivariate analysis was performed using Cox’s proportional hazards model. The data are expressed as the mean ± SEM, and P \ 0.05 was considered statistically significant.
Results TUBA1B Expression in Tumor and Adjacent Nontumor Tissues from HCC Patients
Cell Proliferation Assay and Cell Cycle Analysis Cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay following the manufacturer’s instructions. In brief, cells were seeded at a density of 2 9 104/well into a 96-well cell culture cluster (Corning, Corning, NY, USA) in 100 lL culture medium and incubated overnight. CCK-8 (Dojindo, Kumamoto, Japan) reagents were added to a subset of wells, and cells were incubated for 2 h at 37 °C. The absorbance was recorded with an automated plate reader. For cell cycle analysis, the cells were fixed in 70 % ethanol for 1 h at 4 °C and then incubated with 1 mg/mL RNase A for 30 min at 37 °C. Subsequently, the cells were stained with propidium iodide (50 lg/mL PI) (Becton–Dickinson, San Jose, CA, USA) in PBS plus 0.5 % Tween-20, followed
Immunohistochemical observation indicated that TUBA1B or Ki-67 (a cell proliferation index) was high expressed in most tumor tissues and was low or null expressed in most adjacent nontumor tissues (Fig. 1a–h). Western blot analysis of eight paired tumor and adjacent nontumor tissues further confirmed that TUBA1B expression was higher in tumor tissues than in adjacent nontumor tissues (Fig. 1i). Correlation Between TUBA1B Expression and Clinicopathological Variables The clinicopathological variables and personal data of HCC patients are listed in Table 1. For each variable, patients were divided into high and low TUBA1B expressers according to
123
2716
Dig Dis Sci (2013) 58:2713–2720 Table 1 TUBA1B or Ki67 expression and clinicopathological variables in 114 HCC specimens Variables
Total
TUBA1B Low
23
7
16
Male
91
28
63
Survival analysis was restricted to 96 cases with available complete follow-up data and results of TUBA1B expression. The Kaplan–Meier survival curves of high versus low TUBA1B expressers demonstrated a significant separation (P \ 0.05; Fig. 3), suggesting that high TUBA1B expression was significantly associated with short overall survival. Multivariate analysis using the Cox’s proportional hazards model showed that TUBA1B was an independent prognostic indicator for overall survival in HCC patients (P = 0.004; Table 2).
123
High 0.895
7
16
29
62
14
25
22
53
0.991
B45
39
12
27
[45
75
23
52
0.474
\0.001*
Histological grade
Correlation Between TUBA1B Expression and Survival Rate
Low
P
0.975
Female Age (years)
the immunostaining intensity score (2 and 3: high, 0 and 1: low). Statistics indicated that TUBA1B expression was significantly related to histological grade (P \ 0.001), but not significantly related to other variables, including gender, age, tumor size, metastasis, seronal invasion, HBsAg, serum AFP level, cirrhosis, and vascular invasion. Likewise, patients were also divided into high and low Ki-67 expressers depending on whether the percentage of Ki-67-positive cells was C or \ the median percentage (cutoff value) of 41 %. We also found that the Ki-67 expression was significantly related to histological grade (P = 0.008), but not significantly related to related to all other variables. Very importantly, both TUBA1B and Ki-67 exhibited high expression in most tumor tissues, and there was a positive correlation between TUBA1B and Ki-67 expressions (Fig. 2).
Ki-67
High
Gender
Fig. 1 Representative micrographs, obtained by immunostaining with antibodies against TUBA1B and Ki67, showing TUBA1B (a, b, e, f) and Ki-67 (c, d, g, h) protein expressions in paired tumor (a– d) and nontumor (e–h) tissue samples from HCC patients. Scale bars (a, c, e, g) 40, and (b, d, f, h) 20 lm. b, d, f, h are local magnifications of (a, c, e, g), respectively. Western blot images (i) showing TUBA1B protein expressions in eight pairs of tumor and nontumorous tissue samples from HCC patients. b-actin was used as a protein loading control
P
2
0.008*
I
6
4
II
54
13
41
22
32
III
54
3
51
10
44
Tumor diameter(cm)
4
2
0.206
0.581
B5
59
15
44
20
39
[5
55
20
35
16
39
Distant metastasis
0.656
0.057
Negative
97
29
68
34
63
Positive
17
6
11
2
15
Seronal invasion
0.582
0.199
Negative
72
21
51
26
46
Positive
41
14
27
10
31
7
17
29
61
17
43
19
35
7
14
29
63
HBsAg
0.238
Negative
24
5
19
Positive
90
30
60
AFP
0.775
0.063
B300
60
23
37
[300
54
12
42
Cirrhosis
0.432
0.522
Negative
22
8
14
Positive
92
27
65
Vasular invasion
0.872
0.523
0.063
Negative
80
26
54
30
50
Positive
34
9
25
6
28
\0.001*
Ki-67 expression Negative
36
16
20
Positive
78
4
74
* Significant at P \ 0.05. Statistical analyses were performed by the Pearson v2 test
TUBA1B Expression in Proliferating HCC Cells Western blot analysis indicated that TUBA1B was significantly higher expressed in HCC cell lines, especially in HuH7 cells, than normal liver cells, HL-7702 and chang
Dig Dis Sci (2013) 58:2713–2720
2717 Table 2 The contribution of various potential prognostic factors to survival by Cox regression analysis in 96 HCC specimens
Fig. 2 Relationship between TUBA1B and Ki-67 expression in HCC, as measured using the Spearman rank correlation test
Hazard ratio (HR)
95.0 % confidence interval
P
Gender
1.709
0.963–3.034
0.070
Age (years)
1.168
0.647–2.108
0.606
Tumor grade Tumor size
2.613 1.526
1.309–5.216 0.927–2.514
0.006 0.097
Metastasis
1.374
0.504–3.746
0.535
Serosal invasion
0.963
0.495–1.871
0.911
HBsAg
1.120
0.516–2.433
0.774
AFP
1.211
0.651–2.251
0.546
Cirrhosis
2.031
0.900–4.586
0.088
Vascular invasion
1.163
0.510–2.652
0.720
TUBA1B
2.476a
1.342–4.568
0.004*
Ki-67
2.470a
1.035–5.895
0.042*
* Significant at P \ 0.05. Statistical analyses were performed by the log-rank test a
HR value is given as low versus high expression
Effects of Altered TUBA1B Expression on Cell Growth and Cell Cycle in Human HCC Cell Lines
Fig. 3 Kaplan–Meier survival curves for low versus high TUBA1B expressers in 96 HCC patients
(Fig. 4a). To investigate the change of TUBA1B expression during cell cycle progression of HCC cells, HuH7 cells were subjected to serum starvation and serum refeeding [25]. Flow cytometry data indicated that 66 % of HuH7 cells were arrested at the G0/G1 phase by serum starvation for 72 h, and upon serum refeeding, the cells were released from the G1 phase and they re-entered the S phase (Fig. 4b). Furthermore, western blot analysis showed that, as expected, the expression of TUBA1B or proliferating cell nuclear antigen (PCNA) in HuH7 cells was quite low upon serum starvation for 72 h, and started to increase as early as at 4 h of serum refeeding (Fig. 4c). In other words, TUBA1B expression in HCC cells was gradually increased with the cell cycle progression.
To knockdown endogenous expression of TUBA1B in HuH7 cells, we prepared two siRNA plasmids (siRNA#1 and #2), and transfected them into the cells that highly expressed TUBA1B. Western blot confirmed that transfection with either siRNA#1 or #2 effectively suppressed TUBA1B expression as compared to transfection with pSilencer 4.1-CMV empty vector or no plasmid (control). Cell proliferation was determined by CCK-8 assay at indicated times (0, 24, 48, and 72 h), and the result showed that a significant decrease in the cell proliferation rate was observable due to downregulation of TUBA1B expression in HuH7 cells by siRNA#1 or #2 (Fig. 5b), suggesting that TUBA1B promoted cell proliferation in HCC cells. Flow cytometry was used to observe the cell cycle progression in HuH7 cells at 72 h after transient transfection with or without siRNAs. The percentage of cells in the S phase (1.10 or 6.09 %) after transfected with siRNA#1 or #2 was significantly lower than that (20.01 %) after transfected with pSilencer 4.1-CMV empty vector, whereas the percentage of cells in the G2/M phase (36.47 or 36.46 %) after transfected with siRNA#1 or #2 was significantly higher than that (28.58 %) after transfected with pSilencer 4.1-CMV empty vector (Fig. 5c). These comparisons suggested that downregulation of TUBA1B expression caused HCC cells arrest at the G2 phase.
123
2718
Fig. 4 a Representative western blot images showing TUBA1B protein expressions in 2 normal hepatocyte cell lines (HL-7702 and chang) and 5 HCC cell lines (SMMC-7721, HepG2, BEL-7404, Hep3B, and HuH7). GAPDH was used as a protein loading control. b Flow cytometry data showing the cell cycle progression in HuH7 cells, which were subjected to serum starvation for 72 h (S72h) and then serum refeeding (R) for the designated time periods (R4h–R24h). Data are expressed as mean ± SEM of three independent experiments. *,# P \ 0.01 versus S72 h. c Representative western blot images showing the alterations of TUBA1B or PCNA protein expression in HuH7 cells after they were subjected to serum starvation for 72 h (S72h) and then serum refeeding (R) for the designated time periods (R4h–R24h). b-actin was used as a protein loading control
Correlation Between TUBA1B Expression and Resistance to Paclitaxel in HCC Cells Among tubulin family members, some are the targets of chemotherapeutic drugs while others are chemotherapyresistance factors [9–11, 19, 20]. To investigate the role of TUBA1B in HCC chemotherapy, HuH7 cells, which had been transfected with TUBA1B siRNAs or pSilencer 4.1CMV empty vector, were further treated with different concentrations of paclitaxel, an antimicrotubule agent. The cell colony formation rate was measured by plate colony formation assay. The growth of HuH7 cells transfected with TUBA1B siRNAs was significantly reduced as compared to that transfected with vector or not transfected (control) (Fig. 6), suggesting that decreased TUBA1B expression increased the sensitivity to paclitaxel in HCC cells.
123
Dig Dis Sci (2013) 58:2713–2720
Fig. 5 a Representative western blot images showing TUBA1B protein expression in HuH7 cells after transfected with siRNA#1 and #2. b-actin was used as a protein roading control. b Cell proliferation curves in HuH7 cells transfected with siRNA#1 and #2, pSilencer 4.1CMV empty vector (vector) and no plasmid (control), respectively. The cell proliferation was determined by CCK-8 assay at indicated times (0, 24, 48, and 72 h). Data are expressed as mean ± SEM of three independent experiments. * P \ 0.05 versus control and empty vector. c Flow cytometry data showing the cell cycle progression in HuH7 cells which had been transfected with siRNA#1 and #2, pSilencer 4.1-CMV empty vector (vector) and no plasmid (control), respectively. Data are expressed as mean ± SEM of three independent experiments. *,§,# P \ 0.05 versus control and vector
Discussion Despite significant progress in surveillance and management of HCC, the overall prognosis remains unsatisfactory, and so it is necessary to identify patients with poor prognosis for timely intervention. Further investigation of biomarkers for HCC not only helps define the risk of recurrence but also benefits the development of novel therapeutic targets for HCC patients.
Dig Dis Sci (2013) 58:2713–2720
2719
Fig. 6 Dose responses to paclitaxel of HuH7 cells with TUBA1B knockdown. Equal numbers of HuH7 cells (*100–200) were plated in a 24-well dish. The cells were transfected with siRNA#1 and #2, pSilencer 4.1-CMV empty vector (vector), and no plasmid (control). Then, an increasing concentration of paclitaxel (0–400 ng/mL) was added to allow cell incubation for 7 days, followed by staining with 0.5 % crystal violet
TUBA1B has been known to be associated with the formation of microtubules [7], whereas microtubules play important roles in the regulation of cell proliferation. It is reasonable to speculate that the altered expression of TUBA1B is involved in cancer cell proliferation, thus affecting the pathology of tumors. Although few studies report on the relationship of TUBA1B with HCC, there has been evidence showing that TUBA1B expression is implicated with bad prognosis in mantle cell lymphoma [26]. Therefore, we chose TUBA1B as the subject of this study, and focused on investigating the potential role and prognostic significance of TUBA1B in HCC. The results showed that TUBA1B expression was upregulated in HCC tumor tissues, as compared to that in nontumor liver tissues, suggesting that an increased TUBA1B expression might be associated with the progression of HCC. Subsequently, the relationship between TUBA1B and Ki-67 expressions was examined because Ki-67 is a common index of tumor proliferative activity [27]. We noted that TUBA1B expression was positively related to Ki-67 expression in HCC tissues, indicating that an increased TUBA1B expression might enhance tumor proliferation. The correlation analysis revealed that TUBA1B or Ki-67 expression was significantly associated with histological grade of HCC. This relationship meant that the higher expression of TUBA1B or Ki-67 was linked to the more malignant grade of histological differentiation in HCC. Meanwhile, survival analysis indicated that there was a significant correlation between TUBA1B expression and overall survival. The survival rate of high TUBA1B expressers was lower than that of low TUBA1B expressers, implying that the higher expression of TUBA1B was associated with the poorer prognosis. The multiple Cox’s model further confirmed the prognostic significance of TUBA1B for HCC patients.
Western blot analysis compared TUBA1B expression in HCC cell lines with that in normal liver cells, providing further evidence for the involvement of TUBA1B in the pathology of HCC. In vitro proliferating HCC cells (HuH7) was used as a cell model to investigate the changes of TUBA1B expression during cell cycle progression. We found that TUBA1B expression was upregulated during the G1- to S-phase transition, which is considered a major checkpoint for the cell cycle progression. TUBA1B knockdown in HuH7 cells was found to significantly reduce the cell proliferation rate, and to significantly decrease and increase the cell percentage in the S-phase and G2/M phase, respectively. In addition, we observed that the percentage of HCC cells in the G1 phase was also significantly increased by transfection with siRNA#1 or #2 (62.42 or 57.44 %) as compared to by transfection with pSilencer 4.1-CMV empty vector (51.40 %). This phenomenon may be attributed to inhibition of the cell growth by the interference of microtubule assembly [28, 29]. It has been shown that TUBA3C reduces the sensitivity of ovarian cancer to paclitaxel chemotherapy [20]. Anti-mitotic drugs (for example, paclitaxel) serve as traditional chemotherapeutic agents [11]. Accumulating clinical evidence indicates that acquired resistance or insensitivity to chemotherapeutic drugs represents a big challenge in cancer therapy [2]. As is known, paclitaxel induces anti-tumor effects by disrupting microtubule stabilization and suppressing microtubule dynamics [9–11]. In this study, we found that TUBA1B knockdown could increase the sensitivity of HCC cells to paclitaxel. Inversely, an increased TUBA1B expression in HCC might confer resistance to paclitaxel. To summarize, this study, for the first time, indicated that TUBA1B expression provided important prognostic information in HCC, and TUBA1B expression was associated with the sensitivity to paclitaxel, a chemotherapeutic
123
2720
Dig Dis Sci (2013) 58:2713–2720
drug. Further study is required to elucidate how TUBA1B acts as a useful marker for HCC. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81272708), the Foundation for Talents in Six Fields of Jiangsu Province (No. 2006073), the Health Project of Jiangsu Province (No. H200923), and the Social Development Foundation of Nantong City (No. S2007028 and S2010012). Conflict of interest
14.
15.
16.
None. 17.
References 1. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin. 2005;55:10–30. 2. Cho SJ, Kim YJ, Surh YJ, Kim BM, Lee SK. Ibulocydine is a novel prodrug cdk inhibitor that effectively induces apoptosis in hepatocellular carcinoma cells. J Biol Chem. 2011;286:19662–19671. 3. Simonetti RG, Liberati A, Angiolini C, Pagliaro L. Treatment of hepatocellular carcinoma: a systematic review of randomized controlled trials. Ann Oncol. 1997;8:117–136. 4. Camma` C, Schepis F, Orlando A, et al. Transarterial chemoembolization for unresectable hepatocellular carcinoma: meta-analysis of randomized controlled trials1. Radiology. 2002;224:47–54. 5. Kim ND, Park ES, Kim YH, et al. Structure-based virtual screening of novel tubulin inhibitors and their characterization as anti-mitotic agents. Bioorgan Med Chem. 2010;18:7092–7100. 6. Chen TH, Pan SL, Guh JH, et al. Moscatilin induces apoptosis in human colorectal cancer cells: a crucial role of c-jun nh2-terminal protein kinase activation caused by tubulin depolymerization and DNA damage. Clin Cancer Res. 2008;14:4250–4258. 7. Glotzer M. The 3 ms of central spindle assembly: microtubules, motors and maps. Nat Rev Mol Cell Biol. 2009;10:9–20. 8. Cheung CHA, Wu SY, Lee TR, et al. Cancer cells acquire mitotic drug resistance properties through beta i-tubulin mutations and alterations in the expression of beta-tubulin isotypes. PLoS ONE. 2010;5:e12564. 9. Hage-Sleiman R, Herveau S, Matera EL, Laurier JF, Dumontet C. Tubulin binding cofactor c (tbcc) suppresses tumor growth and enhances chemosensitivity in human breast cancer cells. BMC Cancer. 2010;10:135. 10. Gan PP, McCarroll JA, Byrne FL, Garner J, Kavallaris M. Specific beta-tubulin isotypes can functionally enhance or diminish epothilone b sensitivity in non-small cell lung cancer cells. PLoS ONE. 2011;6:e21717. 11. Tseng CY, Mane JY, Winter P, et al. Research quantitative analysis of the effect of tubulin isotype expression on sensitivity of cancer cell lines to a set of novel colchicine derivatives. Mol Cancer. 2010;9:131. 12. Kohno K, Kikuchi J, Sato S, et al. Vincristine-resistant human cancer kb cell line and increased expression of multidrug-resistance gene. Cancer Sci. 1988;79:1238–1246. 13. Mechetner E, Kyshtoobayeva A, Zonis S, et al. Levels of multidrug resistance (mdr1) p-glycoprotein expression by human
123
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
breast cancer correlate with in vitro resistance to taxol and doxorubicin. Clin Cancer Res. 1998;4:389–398. Hari M, Wang Y, Veeraraghavan S, Cabral F. Mutations in alphaand beta-tubulin that stabilize microtubules and confer resistance to colcemid and vinblastine. Mol Cancer Ther. 2003;2:597–605. Ohishi Y, Oda Y, Basaki Y, et al. Expression of beta-tubulin isotypes in human primary ovarian carcinoma. Gynecol Oncol. 2007;105:586–592. Koh Y, Jang B, Han SW, et al. Expression of class iii beta-tubulin correlates with unfavorable survival outcome in patients with resected non-small cell lung cancer. J Thorac Oncol. 2010;5:320–325. Lobert S, Jefferson B, Morris K. Regulation of beta-tubulin isotypes by micro-rna 100 in mcf7 breast cancer cells. Cytoskeleton (Hoboken). 2011;68:355–362. Kaira K, Serizawa M, Koh Y, et al. Expression of excision repair cross-complementation group 1, breast cancer susceptibility 1, and beta iii-tubulin in thymic epithelial tumors. J Thorac Oncol. 2011;6:606–613. Cho EH, Whipple RA, Matrone MA, Balzer EM, Martin SS. Delocalization of gamma-tubulin due to increased solubility in human breast cancer cell lines. Cancer Biol Ther. 2010;9:66–76. Nikas JB, Boylan KLM, Skubitz APN, Low WC. Mathematical prognostic biomarker models for treatment response and survival in epithelial ovarian cancer. Cancer Inf. 2011;10:233–247. Takano T, Hasegawa Y, Miyauchi A, et al. Overexpression of kalpha1 tubulin mrna in thyroid anaplastic carcinoma. Cancer Lett. 2001;168:51–55. Wang Y, Yang S, Ni Q, et al. Overexpression of forkhead box j2 can decrease the migration of breast cancer cells. J Cell Biochem. 2012;113:2729–2737. Schibler MJ, Cabral F. Taxol-dependent mutants of Chinese hamster ovary cells with alterations in alpha-and beta-tubulin. J Cell Biochem. 1986;102:1522–1531. Kaibori M, Tanigawa N, Kariya S, et al. A prospective randomized controlled trial of preoperative whole-liver chemolipiodolization for hepatocellular carcinoma. Dig Dis Sci. 2012;57: 1404–1412. Ke Q, Ji J, Cheng C, et al. Expression and prognostic role of spy1 as a novel cell cycle protein in hepatocellular carcinoma. Exp Mol Pathol. 2009;87:167–172. Blenk S, Engelmann JC, Pinkert S, et al. Explorative data analysis of MCL reveals gene expression networks implicated in survival and prognosis supported by explorative CGH analysis. BMC Cancer. 2008;8:106. Mann CD, Neal CP, Garcea G, Manson MM, Dennison AR, Berry DP. Prognostic molecular markers in hepatocellular carcinoma: a systematic review. Eur J Cancer. 2007;43:979–992. Sanchez-Perez T, Ortiz-Ferron G, Lopez-Rivas A. Mitotic arrest and jnk-induced proteasomal degradation of flip and mcl-1 are key events in the sensitization of breast tumor cells to trail by antimicrotubule agents. Cell Death Differ. 2009;17:883–894. Kamino H, Moore R, Negishi M. Role of a novel car-induced gene, tuba8, in hepatocellular carcinoma cell lines. Cancer Genet. 2011;204:382–391.
