Mouse recombinant protein HspB6 was purchased from BioChem Technology, Inc. (Shanghai, China). Alexa Fluor^® 488 Annexin V/Dead Cell Apoptosis kit (cat no. V13245) and PE-labeled swine anti-rat IgG polyclonal antibodies were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Rat anti-mouse CD31 (MEC13.3) monoclonal antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Goat anti-mouse VEGF (sc-1836) polyclonal antibodies, goat anti-mouse bFGF (C-18) polyclonal antibodies and goat anti-mouse ICAM-1 (sc-1511) polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

LLC cells, a murine NSCLC cell line, was purchased from Shanghai Biotechnology (Shanghai, China). Cells were maintained in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS. BALB/c mice weighing 20–25 g were kindly supplied by the Immunology Laboratory of Soochow University (Suzhou, China) and were kept in our animal facility under specific pathogen-free conditions. All animal experiments were approved by the Guideline for the Care and Use of Laboratory Animals of the Chinese Medical Academy and the Soochow University Animal Care Committee (Suzhou, China). Animals were kept in groups of five and fed regular laboratory chow and water ad libitum. A 12-h day and night cycle was maintained.

LLC cells were grown in RPMI-1640 culture medium (Invitrogen Life Technologies, Grand Island, NY, USA) containing 10% FBS, 2 mM L-glutamine, 2 mM sodium pyruvate, 20 mM HEPES, 1% non-essential amino acids, 100 μg/ml streptomycin and 100 U/ml penicillin. Cells were maintained at 37°C with 5% CO₂. Cells were progressively passed to larger plates and allowed to reach ~90% confluence. Cells were harvested by trypsinization, washed with HBSS buffer (Invitrogen Life Technologies) three times and resuspended at a density of 1.0×10⁷ cells/ml in serum-free HBSS buffer. LLC cells (1×10⁶) in 100 μl HBSS buffer were injected subcutaneously into 8-week-old male BALB/c mice. Tumor growth was assessed on days 6, 8, 10, 14, 16 and 18 after LLC cells injection by the measurement of two bisecting diameters in each tumor using calipers. The size of the tumor was determined by direct measurement of the tumor dimensions. The volume was calculated according to the equation: V = (LxW²) × 0.5, where V=volume, L=length and W=width (21). On day 14 following tumor implantation, the mice were anesthetized and sacrificed by dislocation of the cervical spine, and the tumor tissues were dissected and weighed.

The mice were sacrificed on day 14 following the implantation of LLC cells, and cervical tissues surrounding tumor masses were excised en bloc. The nearby cervical lymph nodes were removed and their weights were measured immediately.

Total RNA was extracted from LLC cells using an RNeasy Mini kit (Qiagen, Tokyo, Japan). The resultant RNA preparations were further treated with ribonuclease-free deoxyribonuclease (DNase) I (Invitrogen Life Technologies Inc., Gaithersburg, MD, USA) to remove genomic DNA. Total RNA (2 μg) was reverse-transcribed at 42°C for 1 h in 20 μl of reaction mixture containing mouse Moloney leukemia virus reverse transcriptase and hexanucleotide random primers (Qiagen). The PCR solution contained 2 μl of cDNA, the specific primer set (0.2 μM final concentration) and 12.5 μl of SYBR Premix Ex Taq™ (SYBR Premix Ex Taq Perfect Real-Time PCR kit; Takara, Bio, Inc., Shiga, Japan) in a final volume of 25 μl. qPCR was performed using an iCycler iQ Multicolor Real-Time PCR Detection system (170–8740; Bio-Rad, Hercules, CA, USA). The PCR parameters were as follows: Initial denaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The relative gene expression levels were calculated using the 2^−ΔΔCt method, where Ct represents the threshold cycle, and GAPDH was used as a reference gene. The primer sequences are shown in Table I.

Cell lysates from dissected tumor tissues 14 days following injection were prepared. Briefly, protein samples were dissolved in Laemmli buffer (Bio-Rad), boiled for 5–10 min and centrifuged for 2 min at 10,000 × g to remove insoluble materials. Protein (30 μg per lane) was separated by SDS/PAGE (12%; Bio-Rad) and transferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA). The inhibited membranes were probed overnight (4°C) with goat anti-mouse VEGF, bFGF or ICAM-1 antibodies (Santa Cruz Biotechnology, Inc.; sc-1836, 1:100), respectively, and rabbit anti-mouse GAPDH (Santa Cruz Biotechnology, Inc.; N-21, 1:200). Subsequently, the membranes were incubated with secondary horseradish peroxidase-conjugated antibody and immunoreactive bands were visualized using ECL reagent (Pierce Biotechnology, Inc., Rockford, IL, USA). Immunoreactive bands corresponding to target proteins were quantified by Image J analysis (http://rsb.info.nih.gov/ij/download.html) and normalized to those of GAPDH. Blots are representative of at least three experiments.

Tumor tissues dissected from BALB/c mice were minced with scissors and were homogenized using a homogenizer in RPMI-1640 medium. Cell suspensions were then passed over a nylon filter with a 100-μm pore size. The resultant cells were further stained with rat anti-mouse CD31 mAb followed by staining with PE-conjugated swine anti-rat IgG mAb. Fluorescence intensities were determined using a FACSCalibur (Becton-Dickinson, Franklin Lakes, NJ, USA), together with the samples stained with non-immunized swine IgG as an isotype control. In the apoptosis assay, cell suspensions were washed in cold phosphate-buffered saline (PBS) in the absence of an inducing agent. The resultant cells were re-centrifuged and the supernatant was discarded. Cell pellets were resuspended in 100 μl/test annexin-binding buffer. Alexa Fluor^® 488 annexin V (5 μl; component A) and 1 μl 100 μg/ml of PI working solution were added to each 100-μl cell suspension followed by incubation at room temperature for 15 min. Following the incubation period, 400 μl of 1X annexin-binding buffer was added, gently mixed and kept on ice. As soon as possible, the stained cells were analyzed by a flow cytometry calibur.

In order to explore the effects of HspB6 on the migration of LLC cells, wound scratch assays were performed. The assay was simple and inexpensive and the experimental conditions can be easily modified for different purposes. In brief, cells were seeded into six-well plates at a density that, following 24 h of growth, reached 70–80% confluence as a monolayer. The monolayer was gently, slowly and perpendicularly scratched with a new 1-ml pipette tip across the center of the well. The resulting gap distance was therefore equal to the outer diameter of the end of the tip. Following this, the wells were gently washed twice with medium to remove the detached cells. Subsequently, the wells were replenished with fresh medium. Cells were treated with 1 μg/ml of human recombinant HspB6 in experimental wells and PBS in control wells. Cells were grown for an additional 48 h and images of the cells were captured on a microscope (BX43; Olympus Imaging, Shinjuku, Tokyo, Japan) at 0, 12, 24 and 48 h. The gap distance was quantitatively evaluated using Image J software. Each experimental group was repeated multiple times.

The rate of proliferation was determined using Cell Counting Kit 8 (CCK8; Dojindo Laboratories, Kumamoto, Japan). Cells (5×10³ cells per well) were incubated in 96-well plates and maintained in complete medium 24 h following HspB6 stimulation. After 48, 96 and 120 h, 10 μl of sterile CCK8 dye was added to the cells and incubated for 4 h at 37°C. Spectrometric absorbance at a wavelength of 450 nm was measured on an enzyme immunoassay analyzer (ELx-800; Molecular Devices, Sunnyvale, CA, USA) following 4 h of incubation. Experiments were performed at least three times, with six replicate measurements, and data are presented as the mean optical density ± standard deviation.

The means and standard error of the mean were calculated for all parameters determined in the study. Data were analyzed statistically using one-way analysis of variance or two-tailed Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of HspB6 in tumor growth, we firstly grafted LLC cells subcutaneously into HspB6-injected group and control group mice, and evaluated the development of solid tumors surrounding the injection sites. The tumors in the HspB6-injected groups were significantly larger in size than those in the control groups after day 8. On day 14 following implantation, the mean tumor mass in HspB6-injected groups was increased to almost double of that in the control mice (Fig. 1A and B). Flow cytometry examination of the xenografts revealed that the vascularization of tumor tissues was markedly increased by intraperitoneal injection of HspB6 (Fig. 2A and C). CD31 expression of vascular endothelial cells was ~25% higher in tumors grafted in the HspB6-injected groups than those grafted in the control groups (Fig. 2C).

To examine the effects of HspB6 in tumor metastasis, cervical lymph nodes surrounding implanted tumor masses were dissected and weighed. We revealed that the weights of the cervical lymph nodes were markedly increased in HspB6-injected groups implanted with LLC cells in comparison with those of the nodes in the control mice. (Fig. 3A). The subcutaneous implantation of LLC cells in HspB6-injected groups was demonstrated to result in enhanced metastatic growth in the cervical lymph nodes combined with promoting the tumor volume at the implanted site.

To rule out the possibility that the inhibition of cell growth was due to cytotoxic effects, an apoptosis assay was performed. Cells positive for Annexin V-FITC and negative for PI are in the early stage of apoptosis, as shown in the Q4 quadrant, while cells positive for Annexin V-FITC and PI are in the late stages of apoptosis or necrosis, as shown in the Q2 quadrant (22). Thus, the degree of apoptosis correlates with the amount of positive Annexin V-FITC cells. As depicted in Fig. 2B and 2D, there is little binding of Annexin V-FITC on tumor cells implanted in the HspB6 groups compared with the tumor cells implanted in the control mice. The binding for the control mice group ranges from 9.7±2.5 to 12.8±3.1% on day 14. While the binding for the HspB6 group decreased from 6.9±2.7 to 9.5±2.6% on day 14. Combined with these observations, it is clear that the effect of HspB6 on LLC tumors is primarily mediated by reducing apoptotic cell death.

The increased tumor angiogenesis caused large tumor foci in the xenografts of HspB6 groups, however not in the control groups. We also identified that the expression levels of VEGF, bFGF and ICAM-1 protein and mRNA in tumor tissues were markedly higher in HspB6 groups than in the control mice (Fig. 4A and B). The increasing result was confirmed by quantification of the relative abundance of VEGF, bFGF and ICAM-1 to GAPDH using a densitometer (Fig. 4A and C). These results suggest that HspB6 is important in tumor-associated VEGF, bFGF and ICAM-1 production and the accompanying angiogenesis.

In order to further delineate the effects of HspB6 on the biological function of LLCs, we next examined the roles of HspB6 on the migration of LLC cells. LLCs efficiently seal linear scratch wounds following 48 h of culture, and this migratory process was clearly induced by HspB6 supplemented into the culture medium. Cell migration into the wounded area was markedly increased after 24 h in the HspB6 stimulation group (Fig. 5A). These data indicated that an increase in the migration of LLCs following HspB6 stimulation was responsible for the attenuated wound healing response.

To investigate the biological result of HspB6, we also examined the proliferation of LLCs. With the stimulation of HspB6, the proliferation of LLCs was determined by the MTT assay. HspB6 promotes cell proliferation, as determined by the MTT assay, up to 24 h following stimulation with HspB6 (Fig. 5B).