The cysteine protease cathepsin B (Cat B) is important in the progression of tumor cells, however, the function and molecular mechanisms of Cat B in hepatocellular carcinoma (HCC) remain to be elucidated. Our previous study demonstrated that integrin αvβ3 regulated the biological behavior of HCC. The present study demonstrated that Cat B was also important in cell proliferation and apoptosis in HCC. Notably, Cat B was observed to activate the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway to promote HCC proliferation. Furthermore, inhibition of integrin αvβ3 significantly prevented Cat B-induced activation of PI3K/Akt and the progression of HCC. Thus, the results of the present study suggested the presence of a Cat B/integrin αvβ3/PI3K/Akt axis in the regulation of the progression of HCC.
Hepatocellular carcinoma (HCC) is the fifth and sixth most prevalent neoplasm in males and females, respectively, and is the second most frequent cause of cancer-associated mortality in China, following lung cancer (
It is known that the invasion and metastasis of tumor cells are commonly associated with proteases, particularly cysteine protease. There are 11 and 19 members of the cysteine cathepsin family in humans and mice, respectively (
In the present study, Cat B promoted the proliferation and inhibited the apoptosis of HCC cell lines and was identified as an upstream regulator of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Furthermore, the data suggested that integrin αvβ3 was essential in the signal transduction of Cat B into the PI3K/Akt signaling pathway in HCC.
A total of eight pairs of HCC tissues and their adjacent normal tissues were obtained from patients who had undergone surgery at the Department of General Surgery, Qianfoshan Hospital, Shandong University (Jinan, China). The present study was approved by the Hospital Institutional Review Board of Qianfoshan Hospital, Shandong University. Written informed consent was obtained from the patient’s family. The HepG2, SMMC-7721 and BEL-7402 cell lines were obtained from Shandong Province Key Laboratory of General Surgery Center (Jinan, China). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Invitrogen Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St Louis, MO, USA) at 37°C in 5% CO2.
The BEL-7402 cells were transfected with either pcDNA or pcDNA-Cat B using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. The HepG2 and SMMC-7721 cells were transfected with either 40 nM Cat B or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 80% confluence using Geneporter 2 transfection reagent (Genlantis, San Diego, CA, USA) according to the manufacturer’s instructions.
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. A SYBR RT-PCR kit (Takara Bio, Inc., Shiga, Japan) was used for RT-qPCR analysis. The following specific primers were used for RT-qPCR assays: Cat B, forward 5′-CCAGG GAGCA AGACAGAGAC-3′ and reverse 5′-GAGAC TGGCG TTCTC CAAAG-3′ and β-actin, forward 5′-GGCCT CCAAG GAGTA AGACC-3′ and reverse 5′-AGGGG TCTAC ATGGC AACTG-3′. Data from each sample were normalized to the expression of β-actin.
Male, 4-week-old BALB/c nude mice were purchased from the Shanghai Laboratory Animal Company (Shanghai, China). HepG2 cells (1×105) with stable knock down of Cat B and the negative controls were injected subcutaneously under the front legs of the nude mice. The mice were observed over 5 weeks for tumor formation. Subsequently, the mice were sacrificed and, following tumor removal, the wet weights of each tumor were determined.
To prepare the proteins, the treated cell monolayers were rinsed twice using cold phosphate-buffered saline (PBS) and then scraped and transferred into cell lysis buffer containing 50 mM Tris-HCL (pH 7.5), 150 mM NaCl and 0.5% Nonidet P40 (Sigma) with Roche complete EDTA-free protease inhibitor cocktail (Sigma). These suspensions were then centrifuged using a Beckman centrifuge at 10,000 × g for 10 min and the protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal quantities of the proteins were separated using SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) for western blot analysis using primary antibodies against human Cat B (Abcam
Cell viability was assessed using a Cell counting kit-8 (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions.
An annexin-V assay was performed using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit according to the manufacturer’s instructions (BD Biosciences, San Diego, CA, USA). Briefly, 2×105 cells were collected, washed twice with cold PBS, resuspended in 100 μl binding buffer containing Hepes (10 mM), NaOH (pH 7.4), NaCl (140 mM) and CaCl2 (2.5 mM) and incubated with Annexin V-FITC at room temperature for 10 min. This was followed by the addition of 6 μl propidium iodide (PI; 20 μg/ml) for an additional 5 mins. The fluorescent intensities were determined using flow cytometry (Beckman Coulter, Miami, FL, USA).
All data are expressed as the mean ± standard deviation of three or four experiments. Analysis was performed using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.
The expression of Cat B was compared between the HCC tissues and the adjacent normal tissues using RT-qPCR analysis. The results demonstrated that the expression of Cat B was markedly increased in cancer tissues (
To determine the effect of Cat B on HCC cell growth, the BEL-7402 cells were transfected with either the pcDNA3-Cat B or pcDNA3-vector (mock;
The upregulation of Cat B observed in the present study prompted further investigation to determine the effect of its knock down on HCC cell growth. The HepG2 cells were infected with either a lentivirus targeting Cat B (shRNA-Cat B) or a negative control (shRNA-Ctrl). As shown in
To further determine the role of Cat B knockdown, HepG2 cells with stable knock down of Cat B or negative controls were injected subcutaneously under the front legs of the nude mice and tumor growth was closely monitored for 4 weeks. The results demonstrated that the tumor size and weight were markedly reduced following Cat B knockdown compared with the control (
It has been demonstrated that Cat B promotes tumor progression, not only by proteolytic function, but also by a series of signal transduction pathways (
Our previous study demonstrated that the expression of integrin αvβ3 was higher than normal in primary liver cancer and that antisense integrin αvβ3 suppresses the growth of subcutaneously implanted HCC by inhibiting tumor angiogenesis (
Deregulation of cysteine cathepsins functions, including Cat B and Cat L, is associated with a number of disease states, including cancer (
Akt is an important effector kinase, which relays signaling downstream of the PI3K pathway (
Cat B is an extracellular molecule and it has been observed that the interaction of Cat B with urokinase plasminogen activator (uPA), activates the uPA receptor (uPAR) to promote cancer cell invasion and migration, as well as angiogenesis (
This study was supported by the Natural Science Foundation of Shandong Province (grant no. ZR2012HL05).
Cat B is upregulated in HCC tissues and cell lines. (A) Expression of Cat B was determined by reverse transcription quantitative polymerase chain reaction in human HCC tissues and adjacent noncancerous tissues. *P<0.05, compared with normal tissues (n=8). (B) Expression of Cat B was determined using western blot analysis in human HCC tissues and adjacent noncancerous tissues. (C) Expression of Cat B was determined using western blot analysis in HepG2, SMMC-7721 and BEL-7402 cell lines. Cat B, cathepsin B; HCC, hepatocellular carcinoma.
Overexpression of Cat B promotes HCC cell proliferation. (A) BEL-7402 cells were transfected with pcDNA3-Cat B or pcDNA3-vector (mock). Expression of Cat B was determined by western blot analysis. (B) Growth of the BEL-7402 cells transfected with pcDNA3-Cat B or pcDNA3-vector (mock). (C) A BrdU assay was used to detect the proliferative potential of BEL-7402 cells transfected with pcDNA3-Cat B or pcDNA3-vector (mock). (D) Apoptotic rate was determined in BEL-7402 cells transfected with pcDNA3-Cat B or pcDNA3-vector (mock). **P<0.05, compared with Mock. HCC, hepatocellular carcinoma; Cat B, Cathepsin B; BrdU, bromodeoxyuridine.
Cat B knockdown inhibits HCC cell proliferation. (A) Expression of Cat B was determined by reverse transcription quantitative polymerase chain reaction in HepG2 cells infected with shRNA-Cat B or shRNA-Con. (B) Expression of Cat B was determined by western blot analysis in HepG2 cells infected with shRNA-Cat B or shRNA-Con. (C) Growth of HepG2 cells infected with shRNA-Cat B or shRNA-Con. (D) BrdU assay to determine the proliferative potential of HepG2 cells infected with shRNA-Cat B or shRNA-Con. Cat B, cathepsin B; HCC, hepatocellular carcinoma; shRNA-Cat B, lentivirus targeting Cat B; shRNA-Con, negative control; BrdU, bromodeoxyuridine.
Cat B knockdown inhibits the growth of hepatocellular carcinoma
Integrin αvβ3 is essential for Cat B-induced hepatocellular carcinoma progression. (A) Expression levels of T-Akt and p-Akt were determined by western blot analysis in BEL-7402 cells transfected with pcDNA3-Cat B or pcDNA3-vector (mock). (B) Expression levels of T-Akt and p-Akt were determined by western blot analysis in HepG2 cells infected with shRNA-Cat B or shRNA-Con. (C) BEL-7402 cells overexpressing pcDNA3-Cat B were pretreated with integrin αvβ3 inhibiting antibody or isotype-matched control IgG1. Expression levels of T-Akt and p-Akt were determined by western blot analysis. (D) BEL-7402 cells overexpressing pcDNA3-Cat B were pretreated with integrin αvβ3 inhibiting antibody or isotype-matched control IgG1. BrdU assay was used to assess the proliferative potential. (E) BEL-7402 cells overexpressing pcDNA3-Cat B were pretreated with integrin αvβ3 inhibiting antibody or isotype-matched control IgG1. The apoptotic rate was then determined. T-Akt, total Akt; p-Akt, phosphorylated Akt; shRNA-Cat B, lentivirus targeting Cat B; shRNA-Con, negative control; IgG, immunoglobulin G; BrdU, bromodeoxyuridine.