Clin Exp Metastasis (2009) 26:189–195 DOI 10.1007/s10585-008-9230-y

RESEARCH PAPER

Role of PKCb in hepatocellular carcinoma cells migration and invasion in vitro: a potential therapeutic target Kun Guo Æ Yan Li Æ Xiaonan Kang Æ Lu Sun Æ Jiefeng Cui Æ Dongmei Gao Æ Yinkun Liu

Received: 14 January 2008 / Accepted: 26 November 2008 / Published online: 27 December 2008 Ó Springer Science+Business Media B.V. 2008

Abstract Considerable interests have recently been focused on mechanism of human hepatocellular carcinoma (HCC) metastasis—the most fundamental characteristics of HCC and the ultimate cause of most HCC mortality, so screening more potential early prognostic marker and therapeutic target is urgent. In this study, we screened genome of three HCC cell lines with consistently increased metastatic potentials and sharing same genetic background, through DNA microarray and found consecutively up-regulated expression of PKCb in these cell lines compared to others PKCs, which was reconfirmed by real time RT-PCR and western blot analysis. Moreover, it was found, after efficient silence of PKCb by RNAi assay or inhibition of PKCb activity by a specific inhibitor LY317615, migration and invasion of HCC cells significantly decreased. In addition, depletion of PKCb protein significantly reversed the enhancement of PMA-stimulated HCC migration and invasion ability in vitro. All the data suggest a key role of PKCb in HCC motility and PKCb may be a potential therapeutic target. Keywords Hepatocellular carcinoma
DNA microarray
PKCb
Metastasis

K. Guo
Y. Li
J. Cui
D. Gao
Y. Liu (&) Liver Cancer Institute, Zhongshan Hospital, Fudan University, No. 180, Fenglin Rd., Shanghai, China e-mail: [email protected] K. Guo
Y. Li
X. Kang
L. Sun
J. Cui
Y. Liu Research Center For Cancer, Institute of Biomedical Science (IBS), Fudan University, Shanghai 200032, China e-mail: [email protected]

Introduction Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies, with a high mortality rate. Although improvement in survival with available treatments has been reported, HCC mostly engenders a poor prognosis, with a [60% recurrence rate within 5 years after resection due to invasion-related spreading [1, 2]. The poor treatment outcome and dismal prognosis make research on HCC metastasis a high priority. In addition, the development of HCC is expected to follow a multi-step process [3]. Thus, it is critical to identify the molecular mechanisms controlling the invasive and metastatic potential of primary HCC. For a better insight into the mechanisms of human HCC metastasis, a stepwise metastatic human HCC model system, with a similar genetic background including a high metastatic subclone (MHCC97H) and a low metastatic subclone (MHCC97L), was successfully established through in vivo selection of MHCC97, which was derived from the human HCC in nude mice model of LCI-D20 and one even higher metastatic potential cell line HCCLM6 was established from MHCC97H at the authors’ institute [4, 5]. The metastatic process is often characterized as a decathlon in which the rare cancer cell with the right combination of attributes survives to form a metastatic lesion [6]. The challenge then is to discover the minimal set of genes that are functionally necessary for conferring the metastatic phenotype [7]. Although it has been difficult to study this genetic complexity using traditional methods, which are best suited to investigating one gene at a time, but the advent of DNA microarray technology permits the quantitative assessment of complex, multigene expression patterns in cancer. Microarrays yield gene-expression measurements for thousands of genes simultaneously, and

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can be applied to identify a number of candidate genes useful as biomarkers in cancer staging, prediction of recurrence and prognosis, and treatment selection [8, 9]. In this report, we showed that significant up-regulated expression of PKCb in three metastatic HCC cell lines was consistent with these cells metastatic potentials. In addition, further biochemical and functional investigation suggested an important role of PKCb in HCC cells motility and invasion in vitro.

Materials and methods Cell lines Three human HCC cell lines, MHCC97L, MHCC97H and HCCLM6 were cultured at 37°C in 5% CO2 in DMEM medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Hyclone, UT, USA). Briefly, the cells were grown to 90% confluency and harvested by treating with 0.25% trypsin and 0.02% EDTA. Cells were rinsed three times with PBS and centrifuged for further RNA isolation and protein extraction. Genes expression profiling Total RNA was extracted from cells using Trizol reagent (Invitrogen, MD, USA). Further affinity-columns purification of total RNA was performed by NucleoSpinÒ RNA clean-up kit (Macherey-Nagel, Germany). Analysis of gene expression patterns was performed using a Human Oligonucleotide array (Capitalbio Corp, Beijing, China), which contains triplicate spots of oligonucleotide fragments of 909 genes. As a measurement of technical replication, one swap-dye experiment was performed on each biological sample so that a total of six data points were available for a gene on the microarrays. The linear normalization method based on the expression levels of four human housekeeping genes in combination with the yeast external controls was used for data analysis. Normalized data was log transformed and microarray spots in the t-test combined with ratio values 1.5-fold difference were regarded as significant difference of expressed genes. Quantitative real-time RT-PCR To verify the data obtained from microarrays, real time PCR analysis of PKCb was performed by using QuantiTect SYBR Green PCR kit (Qiagen, Valencia,CA, USA) and iCycler IQTM Detection System (Bio-Rad, Reinach, Switzerland) with sequence- specific primer pairs (sense: 50 -GGGCCAAGATCAGTCAG-30 ; antisense: 50 -TCCCC AGCACCATTAGG-30 ). The PCR thermal profile was

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consisted as follows: initiation with a 10-min denaturation at 95°C, followed by 42 cycles of amplification with 10 s of denaturation at 94°C, 20 s of annealing at 57°C, 20–30 s of extension at 72°C, and reading the plate for fluorescence data collection at 78–80°C. After a final extension at 72°C for 5–10 min, a melting curve was performed from 65 to 95°C (1 s hold per 0.2°C increase) to check the specificity of the amplified product. In every assay, runs were independently duplicated and a negative control was included. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense: 50 -ATGACCCCTTCA TTGACC-30 ; antisense: 50 -GAAGA TGGTGATGGGAT TTC-30 ) primers were used to normalize samples for comparison. Results were evaluated with the iCycler IQ real time detection system software (Bio-Rad). Immunoblot analysis Equal amounts of protein extracts in SDS-lysis buffer with phosphatase inhibitor cocktail were subjected to 12% SDS– PAGE analysis and then electrophoretically transferred to PVDF membrane using a Bio-Rad Semi-Dry apparatus. After blocking with blocking buffer (19 TBS, 0.05% Tween-20 with 5% nonfat dry milk or 5% BSA) for 1 h and probing with different antibodies against PKCb (Biosciences Pharmingen, USA), phospho-PKCb (UpstateBiotech., NY, USA) overnight at 4°C, the membrane was incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Enhanced chemiluminescence (Pierce Biotech. Inc., Rockford, USA) system was used for detection. Relative band intensities were determined by quantization of each band with an Imagemaster system. PKC inhibition assays Reactions were done in 50 ll reaction volumes in 96-well polystyrene plates with final conditions as follows: 25 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 5 mM b-glycerophosphate, 0.1 mM NaVO3, 2 mM DTT, 200 lM ATP, 1.5 lM substrate peptide, serial dilutions of LY317615 (0, 0.025, 0.05, 0.1, 1, 2.5, 10 lM), and recombinant human PKCa, PKCb, PKCc, or PKCd enzymes (10, 10, 10, or 50 ng, respectively). Reactions were started with enzyme addition, incubated at room temperature for 15 min, add 50 ll/well stop buffer (50 mM EDTA, pH 8) to stop the reaction. Transfer 25 ll of each reaction to a 96-well streptavidin-coated plate containing 75 ll dH2O/well and incubate at room temperature for 60 min. Then washed and incubated with 100 ll/well phosphor-PKA substrate (RRXS/T) (100G7) rabbit primary antibody at 37°C for 120 min. Washed and added Europium labeled secondary antibody at room temperature for 30 min. After incubated with DELFIAÒ enhancement solution for 5 min, plates were read on time-resolved plate reader.

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RNA interference

MTT assay [11]

Three Small interfering RNAs (siRNA) specifically targeting PKCb was constructed and chemically synthesized. PKC b siRNA-1: 50 -GCCAGUGUUGAUGGCUGGUdT dT-30 (sense), 50 -ACCAGCCAUCAACACUGGCdTdT-30 (antisense); PKCb siRNA-2: 50 -GUGAGGCCAAUGAA GAACUdTdT-30 (sense), 50 -AGUUCUUCAUU GGCCU CACdTdT-30 (antisense); PKCb siRNA-3: 50 -GCCAGUGU UGAUGGCUG GUdTdT-30 (sense), 50 -ACCAGCCAUCA ACACUGGCdTdT-30 (antisense). The siRNAs were evaluated for sequence specificity by a BLAST search and did not show homology to other known genes. An unspecific (non-silencing) siRNA against the target sequence: 50 -UU CUCCGAACGUGUCACGUdTdT-30 (sense), 50 -ACGUGA CACGUUCGGAGAAdTdT-30 (antisense) served as controls. Briefly, 24 h prior to transfection, cells were seeded in 6-well plates at a density of 2 9 105 cells/well. siRNA molecules were transfected with a final concentration of 70 nM for PKCb using the Lipofectamine 2000 reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Lysates were prepared after indicated time in lysis buffer and equal amounts of RNA and protein were subjected to real time-PCR and immunoblot analysis.

Cell viability was assayed by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma) method. Briefly, 5 9 103 cells in 160 ll medium were seeded to each of 96 plate buttom wells in a microtiter plate (3 wells/dose). After cell attachment, specific compounds with prescriptive concentrations were added for the indicated time points. At the end of the treatment, the medium was replaced with 100 ll phenol red-free medium containing 0.5 mg/ml MTT. Cells were incubated for a further 4 h. The MTT medium was then replaced with 200 ll of DMSO, absorbance of each well was measured at 570 nm with a micro-ELISA reader. All MTT assays were repeated three times. Percent cell viability of test samples was determined as %Cell viability = (average OD for test group/average OD for control group) 9 100.

In vitro invasion and migration assay using transwell [10] In vitro invasion assay was performed using 24-well Transwell unit with polycarbonate filters (8 lm pore size; Costar, Acton, MA, USA). Briefly, 3 9 104 cells suspension in DMEM cell culture media with 0.1% BSA were plated on the Matrigel-coated (0.8 lg/ll, 37°C, 2 h; BD Biosciences, San Diego, CA, USA) transwell with or without the presence of LY317615 (0.025 lM), PMA (20 nM) or PKCb siRNA (70 nM). Cells were allowed to invade in 100 ll serum-free DMEM for 20 h through a polycarbonate membrane towards 600 ll NIH-3T3 conditioned medium present in the lower chamber as a source of chemoattractants. Membrane were fixed with methanol for 15 min, and stained with Gimsa for 10 min. Non-invading cells were removed by wiping the upper side of the membrane and cells located on the underside of the filter were counted. For the migration assay, experimental procedures are the same as the in vitro invasion assay described above except that the filter was not coated with Matrigel. Cells were located on the underside of the filter (16 fields/filter) were counted under a light microscope. Three chambers were used per condition. The values obtained were calculated by averaging the total number of cells from three filters.

Statistical analysis Data were expressed as mean ± standard error and analyzed using analysis of variance (ANOVA). Student’s t-test was used in two-group comparisons. The association between the various factors was determined using the Pearson correlation. P \ 0.05 was considered to be statistically significant.

Results Preferentially up-regulated expression of PKCb is identified in metastatic HCC cells To detect genes specifically or preferentially expressed in three metastatic human HCC cell lines, the human oligo microarray including 897 genes was used. A total of 17 genes showed statistically and consecutively up-regulation with ratio values more than 1.5-fold difference and 6 genes showed down-regulation with ratio values \0.5-fold difference. Among 17 genes, PKCb ranked number one with 3.6664-fold difference (MHCC97H/MHCC97L) and 5.1265-fold difference (HCCLM6/MHCC97L). In addition, for others PKC families, although fold difference of PKCa, PKCc, PKCe, and PKCg gene expression in three HCC cells passed t-test, these differences were \1.5-fold; while fold differences of PKCf and PKCh did not pass t-test. So we chose PKCb for further study. The gene expression level of PKCb was further evaluated by quantitative real-time RT-PCR (QRT–PCR) with different paired special primers and it showed fold differences of MHCC97H/MHCC97L and HCCLM6/MHCC97L was

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Fig. 1 The expression of PKCb mRNA and protein was consecutively up-regulated in three HCC cell lines. a Through real time RTPCR analysis, the levels of PKCb mRNA in MHCC97L, MHCC97H and HCCLM6 cells were detected. b These levels of total and activated PKCb were determined by immunoblot analysis of whole cell lysate from three HCC cell lines above using total PKCb antibody and phosphor-specific antibody. The level of GAPDH served as the loading control

3.175 ± 0.562 and 5.624 ± 0.359, respectively Fig. 1a. It was reconfirmed that mRNA levels of the gene was constitutively up-regulated from MHCC97L, MHCC97H to HCCLM6 cells. The analysis of immunoblotting images confirmed that, normalized to the level of GAPDH, level of PKCb expression increased consecutively from MHCC97L to MHCC97H to HCCLM6 and moreover, level of phosphorylated PKCb was also consistently up-regulated in the three cell lines. This result is shown in Fig. 1b. Specific siRNAs efficiently reduce PKCb mRNA and protein expression levels in HCC cells Since PKCb transcription level and protein level were significantly and markedly up-regulated from MHCC97L, MHCC97H to HCCLM6 cells, to obtain insights into whether PKCb was directly related to HCC cells invasion and migration phenotype in vitro, we firstly designed three different siRNAs (siRNA-1, -2, -3) of distinct targeting parts of PKCb mRNA to specifically deplete PKCb in HCCLM6 cell line with the highest metastatic potential among the three cell lines and transfected HCCLM6 with PKCb special siRNAs. Results of QRT-PCR experiments on total RNA obtained from HCCLM6 cell line 2 days after a 4-h transfection with 70 nM of siRNA showed that

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Fig. 2 The level of PKCb mRNA and protein were efficiently inhibited by RNAi assay. HCCLM6 cells were transfected with PKCb siRNA-1, -2 and -3 (70 nM) for 48 h (or not) and then were subjected to real time RT-PCR detection (a) and SDS–PAGE (b). For western blotting analysis, total PKCb antibody and phosphor-specific antibody were used to show pPKCb immunoreactivity. The results represent means of triplicates; *Statistically different from control at P \ 0.05

HCCLM6 cells exposed to siRNA had reduced PKCb mRNA expression level with respect to cells transfected with control siRNA: 0.38 ± 0.07 (siRNA-1), 0.30 ± 0.02 (siRNA-3) and 0.72 ± 0.09(siRNA-2), respectively (Fig. 2a). Results of immunoblotting experiments (Fig. 2b) showed that 2 days after transfection of HCCLM6 with different PKCb siRNAs, PKCb protein expression was reduced by 83 ± 5% in cells with siRNA-1 transfection and 77 ± 3% in cells with siRNA-3 transfection, but little reduced (19 ± 7%) in cells with siRNA-2 transfection, with respect to that observed in cells transfected with control siRNA. Moreover, decreasing level of phosphorylated PKCb (Fig. 2b), which implies PKCb immunoreactivity and enzyme activity was consistent with total PKCb protein in HCC cells. So we chose siRNA-1 and siRNAi-3 for further experiments. Knockdown of PKCb results in decreased HCC cells motility and invasion Efficient inhibition of PKCb expression in metastatic HCC cell line prompted us to demonstrate whether the targeted down-regulation of PKCb has influence on HCC cells invasion and migration phenotype in vitro. We performed an in vitro transwell migration and invasion assay with siRNA-1 and siRNA-3, respectively. The results showed that the number of PKCb-siRNA cells (Migration assay:

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Fig. 3 Depletion of PKCb inhibited HCC cells motility and invasion ability in vitro HCCLM6 cells were pretreated with PKCb siRNA-1 and -3 (70 nM) for 48 h (or not) and then were subjected to migration (a) and invasion (b) assay in vitro for 20 h in the presence of the compound. The results represent means of triplicates; *Statistically different from control at P \ 0.05

35 ± 9 (siRNA-1), 38 ± 7 (siRNA-3); Invasion assay: 28 ± 11 (siRNA-1), 32 ± 12(siRNA-3)) migration and invasion through the filter was markedly lower than the numbers of HCCLM6 without any treatment (Migration assay: 90 ± 15; Invasion assay: 81 ± 10) and control siRNA cells (Migration assay: 84 ± 12; Invasion assay: 74 ± 13) (Fig. 3a, b). Thus, silencing of PKCb gene decreased significantly HCCLM6 cells migration and invasion in vitro. Inhibition of PKCb activity also decreased HCC cells migration and invasion Next, it was reported that as a member of PKC family, intracellular PKCb can be phosphorylated and then activate some signal transduction pathways which control cell proliferation, transformation, survival, apoptosis and so on [12]. So to clarify whether the inhibition of PKCb activity affect HCC cells migration and invasion in vitro, we

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treated HCCLM6 with PKCb special inhibitor LY317615. Due to reports that high concentration LY317615 can inhibit other PKC isozymes activity, we performed firstly a PKC inhibition assay and found 0.025 lM LY317615 inhibited obviously phosphor-PKCb and hardly inhibit phosphorylation of other PKC isoforms (Fig. 4c). The number of PKCb cells with 0.025 lM LY317615 treatment for 1.5 h (Migration assay: 53 ± 12 (siRNA-1), 58 ± 8 (siRNA-3); Invasion assay: 43 ± 10 (siRNA-1), 47 ± 7(siRNA-3)) migrating through the filter was markedly lower than the numbers of HCCLM6 (Migration assay: 93 ± 7; Invasion assay: 85 ± 9) and control siRNA cells (Migration assay: 81 ± 11; Invasion assay: 72 ± 6). To further investigate whether a prominent role of PKCb among PKC isozymes in HCC metastasis, HCCLM6 cells were transfected with PKCb special siRNA then treated by PKC activator PMA. Compared with increasing of migrated and invasive HCCLM6 cells by PMA stimulation, PKCb RNAi-followed-by-PMA-treated HCCLM6 cells motility (HCCLM6: 110 ± 14, Control: 107 ± 17, siRNA1: 57 ± 12, siRNA-3: 50 ± 11) and invasion (HCCLM6: 101 ± 7, Control: 97 ± 12, siRNA-1: 47 ± 9, siRNA-3: 45 ± 13) significantly reduced, while no statistical difference versus inhibitory effect of independently PKCb RNAi-treatment (Fig. 4a, b). This indicated a prominent role of PKCb in HCC cells motility and invasion, compared with other PKC isozymes and increasing of PMAstimulated HCCLM6 cells motility and invasion was mainly through PKCb. In addition, to further ensure whether above results were from toxicity of LY317615 or PMA or absence/deficiency of PKCb on cell survival, as shown in Fig. 4d, cell survival was determined by MTT assay and the decreasing of cell survival rates was not notably shown in LY317615- or PMA-treated HCC cells. Further, PKCb RNAi-treated cells’ survival rate reduced 14%, while decreasing numbers of same treated cells motility and invasion was 53 and 57%. It therefore seems that the main cause of a decreased cells motility and invasive level in HCC cells is absence of PKCb protein rather than PKCb RNAi-induced anti-proliferation.

Discussion The importance of some intracellular protein kinases during tumor cells progression is becoming gradually appreciated. Interactions between tumor cells and malignant phenotype are mediated by various soluble and intrecell kinase molecules, including PKC family. PKC family consists of a number of serine–threonine kinases which are divided into three groups based on their activating factors. Furthermore, functional studies have suggested that PKCs play a role in the carcinogenesis and maintenance/

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Fig. 4 Inhibition of PKCb activity had a down-regulated effect upon HCC motility and invasion ability. For observing effects of PKCb activity inhibition on migrated and invasive ability of PMA-stimulated HCCLM6 cells, HCCLM6 cells were pretreated with 70 nM PKCb siRNA followed by 20 nM PMA (or not), then in vitro migration (a) and invasion assay (b) was performed for 20 h in the presence of the compound. The results represent means of triplicates; *Statistically different from control at P \ 0.05. c PKC inhibition assays with a series concentration LY317615 (0, 0.025, 0.05, 0.1, 1,

2.5, 10 lM) were carried out using HTScanTM PKC kinase assay kit from cell signaling technology, inhibition of PKCa, PKCb, PKCc, or PKCd by various concentration LY317615 were determined through detecting fluorescence values of reaction solutions. The fluorescence value of reaction solution with 0 lM LY317615 served as a percent control for each PKC inhibition assay; d HCCLM6 cells in a 96-well plate was pretreated with 0.025 lM LY317615 or 20 nM PMA or 70 nM PKCb siRNA, Cell survival was determined by MTT assay. The results presented were means of triplicates; bars, ±SE

acceleration of malignant phenotype [13]. Potentiation of malignant phenotype may be mediated by activation of selective PKC isoenzymes or through altered isoenzyme expression profile compared to the originating tissue [14]. In our gene microarray data, significant difference of only PKCb gene was found in metastatic HCC cells, although several other PKC isozymes have been reported in HCC [15]. These differences may result from different HCC cell lines and experimental conditions. For this solution, others PKCs including novel and atypical PKCs will be our research targets in further studies. As a classical PKC isozyme, the role of PKCb in carcinogenesis and progression has been recognized [13]. There is evidence that PKCb can contribute in several ways to tumor formation [16–18]. In addition to direct effects on tumor cells, PKCb is involved in tumor host mechanisms such as inflammation and angiogenesis [19]. It was also demonstrated PKCb knock out mice develop immunodeficiency and show a reduced number of B-lymphocytes. In patients with diffuse large B-cell lymphoma, PKCb is one of the most over-expressed genes and levels of expression are linked to poor prognosis [20]. In this study, the expression of PKCb was investigated in different stepwise

metastatic human hepatocarcinoma cell lines MHCC97L, MHCC97H and HCCLM6. Using cDNA microarray approach, real time-PCR and immunoblotting analysis, we found consecutive overexpression of PKCb protein from lower metastatic MHCC97L to MHCC97H to higher metastatic HCCLM6 cells. This result was consistent with reports on over-expression of PKCb in intestinal cancer development [13]. Taken together, it appeared that high PKCb expression might be associated with HCC metastasis, and be a HCC metastasis-associated gene. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing initiated by short interfering RNA (siRNA) that has become a powerful tool for studying gene function by reverse genetics in mammalian cells [21, 22]. Here, we used RNAi to down-regulate directly the expression of PKCb in HCCLM6 cells. Further observation showed, in contrast to controls, absence/deficiency of PKCb protein led to ability of HCC cells motility and invasion decrease. Meanwhile, inhibition of PKCb activity with special inhibitor LY317615 impeded HCCLM6 cells motility and invasion in vitro. Thus, consistent with reported results, suppression of PKCb expression and activity inhibits cancer cell motility and invasion in HCC cells.

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There are some reports that PKC isozymes are important regulators of cytoskeletal function and PKCb has been shown to interact with the actin cytoskeleton or involved in the process of cellular invasion through activation of a PKCb ? Ras ? PKCi/Rac1 ? Mek signaling pathway [23]. These literatures supported our findings in this study. Moreover, we found PKCb RNAi-followed-by-PMAtreated HCCLM6 cells motility and invasion decreased, compared to PMA-stimulated HCCLM6 cells. These results above implicated that although, as an effective inductor of most PKC isozymes activation including PKCb [24], PMA enhanced HCC cells motility and invasion, absence/deficiency of PKCb sufficiently negated the enhancement. These findings presented here support a crucial and prominent role of PKCb molecule in HCC cells migration and invasion compared to others PKCs, which is perhaps through modulating proliferation and anchorage-independent growth manner in vitro [25]. In summary, our current findings represent an extension of prior findings by us and have important implications for the role of PKCb in HCC cells motility and invasion. These results might at least partially extend the role of PKCb, a potential HCC metastasis promoter. Therefore, PKCb that is related to HCC metastasis might represent an appropriate target for therapeutic approaches of tumor. For example, PKCb inhibitor LY317615 is currently in phase II studies as a single agent in the treatment of gliomas and lymphomas (www.cancer.gov/clinicaltrials). However, future studies will be aimed at exploring the internal mechanism of PKCb in HCC metastasis. Acknowledgments This work was financially supported by national high-tech research and development program of China (2006AA02A308) and the national natural science foundation (30772062).

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