Wu, Huan-Huan
(Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology)
,
Zhang, Shuai
(Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology)
,
Bian, Huan
(Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology)
,
Li, Xiao-Xu
(Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology)
,
Wang, Lin
(Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology)
,
Pu, Yin-Fei
(The Second Dental Center, Peking University School and Hospital of Stomatology)
,
Wang, Yi-Xiang
(Central Laboratory, Peking University School and Hospital of Stomatology)
,
Guo, Chuan-Bin
(Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology)
There are numerous clinical cases indicating that long-term use of bevacizumab may increase the invasiveness of tumors. However, to date, little is known about underlying molecular mechanisms. Therefore, the purpose of our study was to investigate effects of bevacizumab in four cancer cells lines (W...
There are numerous clinical cases indicating that long-term use of bevacizumab may increase the invasiveness of tumors. However, to date, little is known about underlying molecular mechanisms. Therefore, the purpose of our study was to investigate effects of bevacizumab in four cancer cells lines (WSU-HN6, CAL27, Tca83, and HeLa). It was found to promote migration and invasion in the WSU-HN6 and Tca83 cases, while exerting inhibitory effects in CAL27 and HeLa cells. The signal transducer and activator of transcription (STAT) 3 inhibitors niclosamide and S3I-201 inhibited the STAT3 signal pathway, which is activated by bevacizumab. These inhibitors also substantially blocked bevacizumab-induced migration of WSU-HN6 and Tca83 cells. Bevacizumab upregulated interleukin (IL)-6 and phosphorylated (p)-STAT3 expression time-dependently. Therefore, we propose that bevacizumab has differential effects on the migration of different cancer cell lines and promotes migration via the IL-6/STAT3 signaling pathway.
There are numerous clinical cases indicating that long-term use of bevacizumab may increase the invasiveness of tumors. However, to date, little is known about underlying molecular mechanisms. Therefore, the purpose of our study was to investigate effects of bevacizumab in four cancer cells lines (WSU-HN6, CAL27, Tca83, and HeLa). It was found to promote migration and invasion in the WSU-HN6 and Tca83 cases, while exerting inhibitory effects in CAL27 and HeLa cells. The signal transducer and activator of transcription (STAT) 3 inhibitors niclosamide and S3I-201 inhibited the STAT3 signal pathway, which is activated by bevacizumab. These inhibitors also substantially blocked bevacizumab-induced migration of WSU-HN6 and Tca83 cells. Bevacizumab upregulated interleukin (IL)-6 and phosphorylated (p)-STAT3 expression time-dependently. Therefore, we propose that bevacizumab has differential effects on the migration of different cancer cell lines and promotes migration via the IL-6/STAT3 signaling pathway.
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제안 방법
All the PCR reactions were performed in a total volume of 20 μL containing 10 μL of SYBR green PCR master mix, 50 ng cDNA, and 250 nM of each primer.
At the indicated time points following the wounding, the monolayer was photographed at 10× magnification using an inverted microscope and the relative speed of the cell movement was calculated by measuring the distance the cancer cells moved.
However, in this study, we investigated whether bevacizumab has a biological effect on the behavior of tumor cells of epithelial origin. Before we did this study, we firstly tested the VEGF binding activity of bevacizumab by detection of VEGF secretion via ELISA. Our results showed that 100 μg/mL bevacizumab could completely neutralize VEGF secreted by WSU-HN6 and Tca83 cells, which indicates that bevacizumab does have the anti-angiogenic function (Figure 5).
Each experiment was performed in triplicate, and the results were obtained from three independent experiments. All the data were analyzed using the statistics for the social sciences (SPSS) 13.
Four cancer cell lines including three head and neck squamous cell carcinoma (WSU-HN6, CAL27, and Tca83) and one cervical carcinoma (HeLa) were used to investigate the effects of bevacizumab (San Francisco, CA, Genentech, USA) on the migration and invasion of cancer cells. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) or Roswell Park Memorial Institute 1640 medium (RPMI 1640, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37℃ in a 5% CO2 atmosphere.
Therefore, in this study, we examined the direct effects of bevacizumab on different cancer cell lines using the wound healing and transwell invasion assays. Then, we performed a pathway inhibition assay to determine the specific signaling pathway mechanisms that play a dominant role in bevacizumab-induced migration and invasion of cancer cells.
대상 데이터
The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) or Roswell Park Memorial Institute 1640 medium (RPMI 1640, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37℃ in a 5% CO2 atmosphere.
데이터처리
The differences between groups were examined using the Student’s t tests or an analysis of variance (ANOVA), as appropriate.
이론/모형
The relative expressions of the tar¬get genes were calculated using the 2-ΔΔct method.
성능/효과
Finally, we explored the underlying molecule mechanisms using quantitative real-time polymerase chain reaction (qPCR), western blot analysis, and enzyme-linked-immunosorbent assay (ELISA). In conclusion, we found that bevacizumab exerts differential effects on the cell migration of different cancer cell lines, and promotes the migration and invasion of cancer cells primarily via activation of interleukin (IL)-6 / signal transducer and activator of transcription (STAT) 3 pathway.
Next, we detected the effect of bevacizumab on cancer cell migration. Our results clearly showed that, bevacizumab directly affected the migration of tumor cells of epithelial origin but not endothelial cells in vitro, which indicates that it may have other functions rather than anti-angiogenesis. Similar results were obtained in a previous study showing that bevacizumab enhanced the proliferation and invasiveness of glioblastoma cells in vitro (Simon et al.
Therefore, we used a treatment time-course assay to determine the effects of bevacizumab on the expressions of IL-6 and activation of the STAT3 pathway. Our results showed that bevacizumab upregulated IL-6 mRNA and protein levels as well as p-STAT3 protein expression time-dependently. However, the expression of IL-6 mRNA peaked 12 h after treatment.
The results showed that pretreatment with S3I-201 reduced the bevacizumab-induced migration of the WSU-HN6 and Tca83 cells to 11.16±1.59 and 4.33±1.26 μm/h, compared with that observed in the cells exposed to bevacizumab alone (19.23±2.18 and 9.95±1.32μm/h, respectively, P<0.05, Figure 2B).
후속연구
Variations in the genetic backgrounds of the different cancer cells or patients may be responsible for the apparent discrepancy in the responses to bevacizumab. However, the underlying mechanisms of these effects currently remain largely unknown, and further studies are required to elucidate them.
, 2014). Therefore, further studies are required to provide a complete profile of the molecular mechanisms mediating bevacizumab-induced cancer cell migration, including the involvement of the STAT3 pathway.
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