Adenoviral vector-mediated ectopic expression of CACNA2D2 gene inhibited NSCLC cell growth in vitro. (a) Expression of the CACNA2D2 gene in Ad-CACNA2D2 (CACN)-transduced NSCLC cells by RT–PCR analysis. Total RNAs were prepared from H1299, A549, H460, and H358 cells transduced by the Ad-CACNA2D2 vector for 48 h at MOIs of 1000 and 2500 vp/c, respectively, and the RNA prepared from cells transfected with a CACNA2D2-expressing plasmid DNA (pLJ58) was used as a positive control. The RNA samples were treated with DNAse prior to the RT reaction. (b) Western blot analysis of expression of CACNA2D2 protein. The crude protein lysates were prepared from NSCLC cells treated in the same way as described for RNA sample preparation in a. The rabbit anti-CACNA2D2 polyclonal antibodies were used for blotting. (c) XTT assay shows the effect of ectopic expression of CACNA2D2 on tumor cell viability. Cells from NSCLC cell lines A549, H460, H1299, and H358 were tranduced with Ad-CACNA2D2 vectors (CACN) at varied MOIs: 2500 for A549, 4000 for H460, 1000 for H1299, and 2000 vp/c for H358 cells. Untreated (PBS), Ad-EV (EV) treated, and Ad-LacZ(LacZ)-treated cells were used as negative controls and Adp53(p53)-treated cells as a positive control, at the same MOIs as CACN-treated cells for each cell line. Cell viability was calculated relative to that of untreated (PBS) controls. Differences were significant in the CACN-transduced cells compared to the untreated (PBS) control cells (P =0.021 in H1299, P<0.0001 in H358, H460, and A549 cells) and to the Ad-LacZ-transduced cells (P<0.0001 in H358, H460, and A549 cells) after 5 days of transduction. Differences between CACN-treated cells and controls were not significant in the H1299 cell line. (d) Effects of intratumoral administration of CACN on growth of human lung cancer H460 subcutaneous tumors in nu/nu mice. Results were reported as the mean±s.d. in five to 10 mice for each treatment group. Tumor volumes were normalized by the percentage increase of tumor sizes after treatment relative to those at the beginning of the treatment in each group. Mean tumor volumes±s.e. from these experiments are shown. The differences of the tumor volumes in the CACN-treated mice versus the PBS- and Ad-LacZ-treated controls were significant (P<0.0001 and 0.015, respectively)

Apoptosis is induced by adenoviral vector-mediated expression of CACNA2D2 in vitro. NSCLC cell lines A549 (a), H460 (b), H1299 (c), and H358 (d) were tranduced with Ad-CACNA2D2 vectors (CACN) at varied MOIs for each line as shown in Figure 1. Untreated (PBS), Ad-EV (EV)-treated, and Ad-LacZ (LacZ)-treated cells were used as negative controls and Ad-p53(p53)-treated as a positive control. The percentage of apoptosis (TUNEL-positive) in cells transduced for 2 or 5 days, respectively, was determined by FACS analysis. Induction of apoptosis was significant in CACN-treated A549, H460, and H358 cells compared to those in PBS-treated (P =0.0004, 0.0321, and 0.0003, respectively) and to those in Ad-LacZ-treated (P =0.021, 0.048, and 0.027, respectively) controls 5 days after transduction. Differences in induction of apoptosis were not significant (P>0.05) between CACN-treated cells and controls in A549, H460, and H358 cell lines at 48 h post-transduction and in H1299 at both 48 and 120 h post-transduction

Ectopic expression of CACNA2D2 increased the level of the intracellular free calcium. (a) Fluorescence image analysis of free cytosolic Ca²⁺ in Ad-CACNA2D2 (CACN)-transduced H460 and A549 cells by Fluo 3-AM staining. Images of the CACN-treated H460 cells at an MOI of 4000 vp/c (c) and A549 cells (g) at an MOI of 2500 vp/c, untreated (PBS) (a and e) and Adp53 (p53)-treated cells (b and f), and ionomycin-treated control cells (d and h) at 48 h post-treatment are shown. (b) Free Ca²⁺-specific fluorescence emission was quantified by FACS analysis with Fluo 3-AM staining. Cells were treated as described in panel. (a) The differences of free intracellular Ca²⁺ are expressed semiquantitatively through the differences in relative fluorescence. The increase in free cytosolic Ca²⁺ was significant in the CACN-treated H460 (P = 0.015 and 0.032) and A549 cells (P =0.001 and 0.002), but not significant in H1299 cells (P =0.785 and 0.865) compared with the increase seen with untreated (PBS) and p53-treated controls

Ectopic expression of CACNA2D2 reduced the mitochondria membrane potential. (a) Changes in the mitochondria membrane potential in Ad-CACNA2D2-transduced A549 cells are revealed by FACS analysis with JC-1 staining. A549 cells were transduced with adenoviral vectors for 6, 24, 48, and 72 h at an MOI of 2500 vp/c. Fluorescence emission of red at 590 nm (indicating high membrane potential and aggregation of JC-1 dye) and green at 530 nm (indicating the collapse of membrane potential and the monomer of JC-1 dye) were measured by FACS. CACN (n, o, p, q)- and p53 (j, k, l, m)-transduced cells showed mitochondria membrane depolarization as evidenced by the fluorescence emission shift to the longer wavelength of green. PBS (a, b, c, d)- and LacZ (e, f, g, h)-transduced cells did not show a marked shift to the right. (b) Relative green fluorescence was higher in CACN-transduced A549, H460, and H1299 cells than in untreated (PBS) control cell. The decrease of the mitochondria membrane potential or depolarization is significant in the CACN-transduced A549 cells (P = 0.002) and H460 (P = 0.005) but not in H1299 cells after 48 h of transduction or in the untreated (PBS) control cells

Western blotting was used to analyse cyt c release from mitochondria to cytosol in Ad-CACNA2D2-transduced cells. (a) Western blot of cyt c in CACN-transduced A549 and H460 cells. Cells were transduced with adenoviral vectors at an MOI of 2500 vp/c for A549 and MOI of 4000 vp/c for H460 for 48 h, and untreated (PBS) (lanes 1 and 4) and Ad-LacZ (LacZ)-transduced (lanes 2 and 5) cells were used as controls. Immunoblotting for cyt c was performed in fractionated lysates of mitochondria (lanes 1–6) and cytosol (lanes 7–12). Immunoblots of COX IV (I) were used as a mitochondria enzyme marker and β-actin as an internal loading control. (b) Time course of cyt c release in Ad-CACNA2D2-transduced A549 cells. (c) Immunofluorescence image analysis of the subcellular rearrangement of mitochondria and translocation of cyt c in Ad-CACNA2D2-transduced A549 cells. Mitochondria were probed with an FITC-labeled cyt c antibody (green) and the nucleus was counterstained with PI (red). (d) Timetable of induction of apoptosis in Ad-CACNA2D2-transduced A459 cells by FACS analysis with PI staining for DNA content. The percentage of induction of apoptosis was indicated by the increase of the SubG₀-G₁ cell populations (shown by a bar in each plot)

Downstream caspase 3 and PARP are activated by ectopic expression of the CACNA2D2 gene. (a) Western blot analysis of caspase 3. The whole cell lysate was prepared from Ad-CACNA2D2 (CACN)-transduced H460 cells (lanes 1–3) at an MOI of 4000 vp/c and A549 (lanes 5 and 6) cells at an MOI of 2500 vp/c after 72 h of transduction, and untreated (PBS) and Ad-LacZ (LacZ)- or Ad-p53 (p53)-transduced cells were used as controls. The cleaved procaspase 3 was indicated by an arrow. (b) Western blot analysis of PARP. Cells were transduced by Ad-CACNA2D2 (CACN) at the same MOI as described in a for each cell line. The cleaved PARP is indicated by the immunoblotting complexes of about 85 kDa. β-actin was used as an internal loading control