Insufficient Radiofrequency Ablation Promotes Angiogenesis of Residual Hepatocellular Carcinoma Via HIF-1α/VEGFA

Background: The mechanism of rapid growth of the residual tumor after radiofrequency (RF) ablation is poorly understood. In this study, we investigated the effect of hyperthermia on HepG2 cells and generated a subline with enhanced viability and dys-regulated angiogenesis in vivo, which was used as a model to further determine the molecular mechanism of the rapid growth of residual HCC after RF ablation. Methodology/Principal Findings: Heat treatment was used to establish sublines of HepG2 cells. A subline (HepG2 k) with a relatively higher viability and significant heat tolerance was selected. The cellular protein levels of VEGFA, HIF-1α and p-Akt, VEGFA mRNA and secreted VEGFA were measured, and all of these were up-regulated in this subline compared to parental HepG2 cells. HIF-1α inhibitor YC-1 and VEGFA siRNA inhibited the high viability of the subline. The conditioned media from the subline exerted stronger pro-angiogenic effects. Bevacizumab, VEGFA siRNA and YC-1 inhibited proangiogenic effects of the conditioned media of HepG2 k cells and abolished the difference between parental HepG2 cells and HepG2 k cells. For in vivo studies, a nude mouse model was used, and the efficacy of bavacizumab was determined. HepG2 k tumor had stronger pro-angiogenic effects than parental HepG2 tumor. Bevacizumab could inhibit the tumor growth and angiogenesis, and also eliminate the difference in tumor growth and angiogenesis between parental HepG2 tumor and HepG2 k tumor in vivo. Conclusions/Significance: The angiogenesis induced by HIF1α/VEGFA produced by altered cells after hyperthermia treatment may play an important role in the rapid growth of residual HCC after RF ablation. Bevacizumab may be a good candidate drug for preventing and treating the process

Study on Evaluating the Sustainability of Innovative Products

In the process of product innovations, it is necessary to evaluate the sustainability of innovative products in order to meet the demands of sustainable development. So, the calculation process for evaluating the sustainability of innovative products is proposed in this paper. Firstly, the performances of the innovative products in social, economic, and environmental aspects are calculated based on the concepts proposed in this paper, including social matching degree, economic feasible degree, and environmental improving rate. Then, the vector-cosine method in the three-dimensional space is proposed to calculate the sustainability integrated in the three aspects, which regards innovative products as the vectors. Both the size and direction of the vectors are considered to calculate the sustainability. Finally, a case study of the cloud rail made by BYD Company in China is provided to illustrate the validity of the proposed calculation process

The enhanced pro-angiogenic ability of HepG2 k is in a HIF-1α/VEGFA dependent manner <i>in vitro</i>.

<p>(A) HUVECs were treated with conditioned media from parental HepG2 or HepG2 k cells with addition of bevacizumab (0.5 mg/ml) or control IgG for 24 h. Cell viability were quantified by MTT assay. (B–C) HUVEC migration <i>in vitro</i> in response to conditioned media from HepG2 k or parental HepG2 cells with addition of bevacizumab (0.5 mg/ml) or control IgG was assayed after 12 h. The number of migrated cells was quantified by counting 10 random fields at ×100 magnification. (D–E) HUVEC tube formation in response to conditioned media from HepG2 k or parental HepG2 cells with addition of bevacizumab (0.5 mg/ml) or control IgG after 20 h was assayed. The length of tube was evaluated by counting 10 random fields at ×100 magnification. (F–H) The parental HepG2 and HepG2 k cells were treated with or without VEGFA siRNA or 5 µM YC-1, and the conditioned media was collected. The effect of various conditioned media on HUVEC 24 h viability was quantified by MTT assay. The effect of various conditioned media on HUVEC migration for 12 h was assayed. The number of migrated cells was quantified by counting 10 random fields at ×100 magnification. The effect of various conditioned media on HUVEC tube formation for 20 h was assayed. The tube length was evaluated by counting 10 random fields at ×100 magnification. *, <i>P</i><0.05, ***, <i>P</i><0.001 versus parental HepG2 cell control; ^##, <i>P</i><0.01, ^###, <i>P</i><0.001 versus HepG2 k cells control; ns, no significance.</p

Bevacizumab impaired the tumor growth and angiogenesis in tumor-bearing mice <i>in vivo</i>.

<p>2×10⁶ HepG2 k or parental HepG2 cells in cells in 200 µl phosphate buffered saline were injected by subcutaneous injection to obtain s. c. tumors. (A) Average tumor volume is shown for the HepG2 tumors. Parental HepG2 tumor with control IgG (n = 5), Parental HepG2 tumor with Bevacizumab (n = 5), HepG2 k tumor with control IgG (n = 5) and HepG2 k tumor with Bevacizumab (n = 5). **, <i>P</i><0.01. (B) Mice were killed after 28 days implantation and the tumor tissues were removed and weighed. *, <i>P</i><0.05. (C–D) Twenty-eight days after implantation, the numbers of new microvessels marked with CD34 (arrow heads) in the subcutaneous tumors were quantified by performing new vessel counts of 10 random fields at ×400 magnification. ^*, <i>P</i><0.05, ^**, <i>P</i><0.01 versus parental HepG2 tumor respectively; ^###, <i>P</i><0.001 versus HepG2 k tumor. (E) Immunohistochemistry analysis of the expression of VEGF in implanted tumors.</p

HepG2 k cells express elevated level of VEGFA.

<p>(A) Expression of p-Akt, HIF-1α and VEGFA in parental HepG2 and HepG2 k cells were detected by western blot analysis. (B) The mRNA expression of VEGFA in parental HepG2 and HepG2 k cells was assayed using real time PCR. *, <i>P</i><0.05. (C) VEGFA concentration in conditioned media from parental HepG2 and HepG2 k cells was measured by ELISA analysis. **, <i>P</i><0.01. (D) The expression of HIF-1α and VEGFA in parental HepG2 and HepG2 k cells treated with or without LY294002 (20 µM) for 24 h were analyzed by western blot analysis. (E) VEGFA concentration in conditioned media was detected by ELISA analysis. ***, <i>P</i><0.001 versus parental HepG2 cells control respectively; ^###, <i>P</i><0.001 versus HepG2 k cells; ns, no significance.</p

The viability of HepG2 cells and sublines derived from HepG2 cells after hyperthermia.

<p>(A) HepG2 cells were cultured after 47°C heat treatment. The 24 h, 48 h and 72 h cell viability of HepG2 cells with or without 47°C heat treatment were measured using MTT assay. (B) Twenty-four sublines were established after 47°C heat treatment for 10 min as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037266#s2" target="_blank">method</a>. The 24 h, 48 h, and 72 h viability was evaluated by MTT assay after 24 sublines were established. par, parental HepG2 cells; a–x, sublines derived from the HepG2 cells. (C) The 24 h, 48 h and 72 h viability of representative sublines of HepG2 cells were evaluated by MTT assay. (D) Parental HepG2 and HepG2 k cells were treated with 49°C or 50°C 10 min. The 4 h, 12 h, 24 h and 48 h cell viability were measured by MTT assay. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i> <0.001. Data are the representative results of three independent experiments. The coefficients of variation (CV) of all assays were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037266#pone.0037266.s001" target="_blank">Supporting Information S1</a>.</p

YC-1 and VEGFA siRNA inhibit HepG2 cell viability.

<p>The parental HepG2 and HepG2 k cells were treated with or without YC-1(5 µM) or transfected with or without VEGFA siRNA. (A) The expression of HIF-1α and VEGFA was detected by western blot analysis. (B) VEGFA concentration in conditioned media was detected by ELISA analysis. **, <i>P</i><0.01, ***, <i>P</i><0.001 versus parental HepG2 cells control respectively; ^###, <i>P</i><0.001 versus HepG2 k cells. (C) Parental HepG2 and HepG2 k cells were transfected with or without VEGFA siRNA for 24 h, 48 h, and 72 h, cell viability was measured by MTT assay. ***, <i>P</i><0.01; ns, no significance. (D) Parental HepG2 and HepG2 k cells were treated with or without YC-1 (5 µM) for 24 h, 48 h, and 72 h, cell viability was measured by MTT assay. ***, <i>P</i><0.001; ns, no significance.</p