It has been reported that kallikrein 11 (KLK11) is crucially involved in the development and progression of various types of cancer. However, the molecular mechanisms that underlie the involvement of KLK11 in aberrant colorectal cancer (CRC) cell growth remain largely unclear. The aim of the present study was to investigate the role of KLK11 and the effects of KLK11 on oxaliplatin (L-OHP) chemosensitivity by knocking down KLK11 in LOVO and HCT-8 cells. Loss-of-function assays revealed KLK11 inhibition significantly inhibited growth and induced apoptosis of CRC cells
Colorectal cancer (CRC) is among the most common malignancies and the second leading cause of cancer-associated mortality, following lung cancer (
Human kallikrein 11 (KLK11) is a member of the human
The aim of the present study was to investigate the role of KLK11 in human CRC. Additionally, the potential use of shRNA-mediated KLK11 gene knockdown associated with apoptosis and drug resistance were further examined.
Two human-derived CRC cell lines LOVO (CCL-229) and HCT-8 (CCL-244) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured with RPMI-1640 (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in 5% CO2 at 37°C.
Total RNA from cells was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's directions. Then, 1 µg total RNA was used for reverse transcription reaction using SuperScript III reverse transcriptase (Invitrogen). qPCR was performed using an ABI 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA), and the mRNA expression of human KLK11 and β-actin was evaluated using a LightCycler Fast Start DNA Master SYBR Green I kit (Roche Diagnostics GmbH, Mannheim, Germany). PCR amplification was performed by denaturation at 95°C for 10 min, annealing and extension at 60°C for 60 sec for 40 cycles. RT-qPCR analysis was performed using the following primers: KLK11 forward: 5′-GTTCGAGAAGACGCGGCTAC-3′; KLK11 reverse: 5′-GGTGGGAGAGGTGAGTGAC-3′. β-actin forward: 5-CCA ACC GCG AGA AGA TGA-3′; β-actin reverse: 5′-CCAGAGGCGTACAGGGATAG-3′. The relative expression level of KLK11 was calculated using the ΔΔCq method (
In order to silencing KLK11, the short hairpin RNA (shRNA) were generated by ligating synthetic oligonucleotides (Invitrogen) against the target genes into the
Cell viability was quantified using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (
For drug sensitivity, cells were plated in 96-well plates at 5×104 cells per well, followed by treatment with 0, 5 or 10 µmol/lL-OHP for 24 h. The optical density was then measured and the cell viability was calculated.
Apoptosis detection was performed using an Annexin V Apoptosis Detection kit I (BD Biosciences, Franklin Lakes, NJ, USA). In brief, cells were collected and washed with phosphate-buffered saline (PBS). Then, 5 µl annexin V and propidium iodide was added to the cell suspension and incubated at room temperature in the dark for 30 min. The volume was then made up to 500 µl and the cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences).
The activity of caspase-3 was measured using a Caspase-3 Assay kit (Abnova Corporation, Taipei, Taiwan) according to the manufacturer's instructions. In brief, 5×106 cells were harvested, resuspended in 50 µl chilled cell lysis buffer and incubated on ice for 10 min. Then, 50 µl 2.0X Reaction Buffer was added to each sample, along with 5 µl DEVD-pNA (4 mM) substrate and incubated for 2 h at 37°C. The optical density was measured at 405 nm using a microplate reader (Bio-Rad Laboratories, Inc.).
Cell lysates were prepared in a buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% NP-40 (v/v) and 150 mM NaCl, supplemented with a mixture of complete protease inhibitors (Roche Diagnostics, Basel, Switzerland). Equal quantities of protein (40 µg) were then separated on 10% SDS-PAGE and blotted onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). Blocking was performed at room temperature using Tris-buffered saline with 0.1% Tween-20 (TBST; J&K Chemical Ltd., Shanghai, China) containing 5% non-fat milk for 1 h. The membrane was then incubated with primary mouse monoclonal KLK11 antibody (sc-20387; 1:500) and rabbit polyclonal β-actin (sc-47778; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Bcl-2 (#2872; 1:1,000) and Bax (#2772; 1:1,000) antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) in TBST at 4°C overnight and with the appropriate horseradish peroxidase-conjugated secondary antibody (CW0103M; 1:3000; CWbiotech, Beijing, China) for 1 h at room temperature. Specific antibody binding was detected using an ECL system (GE Healthcare, Piscataway, NJ, USA).
Data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). Statistical significance was considered to be indicated by P<0.05.
A previous study has shown that the expression of KLK11 was upregulated in colorectal tumors (
To determine the biological function of KLK11 in CRC progression, MTT assays were used to examine the proliferative ability of CRC cells. As shown in
There is considerable evidence indicating that apoptosis has a close association with cell growth (
To examine the mechanism underlying the inhibition of cell growth, the expression of Bcl-2 and Bax, two important proteins of the apoptosis signaling pathway (
Our previous study has shown that dysregulation of KLK11 expression had an association with FOLFOX4 chemotherapy in human CRC cells (
Overexpression of KLK11 is a general feature in numerous human malignancies including CRC, and the overexpression is often correlated with malignant behavior (
KLK11 is a member of the KLK family, which are dysregulated in multiple tumors (
In order to determine the underlying mechanisms by which KLK11 is involved in cell growth, the present study analyzed apoptosis signaling in CRC cells. Bcl-2 and Bax, two crucial regulatory proteins that play important roles in the induction of apoptosis have been reported to regulate cancer growth (
There is considerable evidence supporting the hypothesis that mechanisms involved in resistance to chemotherapy correlate with apoptosis (
Based on these data, we conclude that knockdown of KLK11 could inhibit cell proliferation, induce apoptosis and increase the sensitivity of CRC cells to L-OHP
Establishment of cell lines that stably knockdown KLK11. Expression of (A) KLK11 mRNA and (B) KLK11 protein in LOVO cells and (C) KLK11 mRNA and (D) KLK11 protein in HCT-8 cells. mRNA levels were detected by reverse transcription-quantitative polymerase chain reaction, and protein levels by western blot analysis. β-actin was used as a loading control. *P<0.05, **P<0.01 vs. SCR. KLK11, kallikrein 11; SCR, short hairpin RNA control.
KLK11 shRNA inhibited cell proliferation and enhanced apoptosis. Proliferation levels of (A) LOVO and (B) HCT-8 cells after KLK11 silencing analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (C) Flow cytometric analysis of apoptosis in LOVO and HCT-8 cells transfected with SCR or shKLK11. (D) Quantification of the apoptotic cells. *P<0.05, **P<0.01 vs. SCR. SCR, short hairpin RNA control; KLK11, kallikrein 11.
Effect of KLK11 silencing on the apoptosis signaling pathway. (A) Western blot analysis of Bcl-2 and Bax protein expression in LOVO and HCT-8 cells transfected with SCR or shKLK11. β-actin was used as a loading control. (B) Relative caspase-3 activity was determined in LOVO and HCT-8 cells transfected with SCR or shKLK11. **P<0.01 vs. SCR. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated protein X; SCR, short hairpin RNA control; KLK11, kallikrein 11.
KLK11 silencing modulates the sensitivity to L-OHP. Viability of KLK11 knockdown (A) LOVO and (B) HCT-8 cells treated with the indicated concentrations of L-OHP for 24 h. (C) The effect of L-OHP on apoptotic levels of the indicated cells analyzed by flow cytometry. (D) Quantification of the apoptotic cells. *P<0.05, **P<0.01 vs. control. KLK11, kallikrein 11; L-OHP, oxaliplatin.