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01.12.2014 | Research | Ausgabe 1/2014 Open Access

Journal of Ovarian Research 1/2014

Gemcitabine as a molecular targeting agent that blocks the Akt cascade in platinum-resistant ovarian cancer

Zeitschrift:
Journal of Ovarian Research > Ausgabe 1/2014
Autoren:
Hiroshi Kawaguchi, Yoshito Terai, Akiko Tanabe, Hiroshi Sasaki, Masaaki Takai, Satoe Fujiwara, Keisuke Ashihara, Yoshimichi Tanaka, Tomohito Tanaka, Satoshi Tsunetoh, Masanori Kanemura, Masahide Ohmichi
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1757-2215-7-38) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

HK, AT, KA and TT performed the MTS assay, invasion assay, Western blot analysis, zymographic assay and statistical analysis. HT, SF and HS conducted the cancer cell cultures and the extraction of mRNA for RT-PCR and protein for the Western blot analysis, performed part of the gene expression experiments and cultured the cells. HK and ST provided the athymic nude mice, performed the tumor cell injections and collected the peritoneal disseminated tumors. YT participated in the conception and design of the study and drafted the manuscript. HK, AT and YT participated in designing the study. YoT and MK evaluated the histology of the tumor samples and the immunohistochemical staining. MO contributed methodological knowledge and participated in designing the study. All authors read and approved the final manuscript.

Abstract

Background

Gemcitabine (2′, 2′ –difluorodeoxycytidine) is one of many nonplatinum drugs that exhibit activity in recurrent, platinum-resistant ovarian cancer. However, the molecular mechanisms by which Gemcitabine treatment inhibits the proliferation of platinum-resistant ovarian cancer cells still remain unclear. We investigated whether Gemcitabine increases the efficacy of Cisplatin in platinum-resistant ovarian cancer models in vitro and in vivo.

Methods

We used Cisplatin-resistant Caov-3 cells, A2780CP cells and Cisplatin-sensitive A2780 cells to examine the sensitivity of the cell viability of Cisplatin and Gemcitabine using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and the sensitivity of the invasive activity of Cisplatin and Gemcitabine using an invasion assay with Matrigel. We examined the Akt kinase activity and matrix metalloproteinase 9 (MMP9) expression following Cisplatin and Gemcitabine treatment using a Western blot analysis and the mRNA expression of vascular endothelial growth factor (VEGF) using semi-quantitative RT-PCR. Moreover, we evaluated the effects of Cisplatin and Gemcitabine on the intra-abdominal dissemination of ovarian cancer in vivo.

Results

Gemcitabine significantly inhibited Cisplatin-induced Akt activation in the Caov-3 and A2780CP cells, but not in the A2780 cells. In the presence of Gemcitabine, Cisplatin-induced growth inhibition and apoptosis were significantly enhanced in the Caov-3 and A2780CP cells. Co-treatment with Cisplatin and Gemcitabine almost completely inhibited invasion of both types of cells through the Matrigel; however, neither Cisplatin nor Gemcitabine alone inhibited the invasion of both types of cells. Gemcitabine inhibited not only the Cisplatin-induced activation of Akt, but also the MMP9 and mRNA expression of VEGF. Moreover, treatment with Gemcitabine increased the efficacy of Cisplatin-induced growth inhibition of the intra-abdominal dissemination and production of ascites in the athymic nude mice inoculated with Caov-3 cells.

Conclusions

We herein demonstrated that Gemcitabine inhibits the Akt kinase activity and angiogenetic activity following treatment with Cisplatin in platinum-resistant ovarian cancer cells. These results provide a rationale for using Gemcitabine in clinical regimens containing molecular targeting agents against platinum-resistant ovarian cancers.
Zusatzmaterial
Additional file 1: Figure S1: Gemcitabine sensitivity in Caov-3 (A), A2780CP (B) and A2780 (C) cells. The cells were treated with Gemcitabine at various concentrations for 24 hours. The number of viable cells was assessed using an MTS assay, as described in the Materials and Methods section. **p < 0.01. , *p < 0.05. (TIFF 33 KB)
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Additional file 2: Figure S2: Effect of combination treatment with Cisplatin and Gemcitabine on the VEGF mRNA expression in A2780CP (A) and A2780 (B) cells. The cells were treated with various combinations of 100 nM of Gemcitabine and 200 μM of Cisplatin for six hours. Total RNA was isolated and reverse transcribed, and the resulting cDNA was used in PCR for the semi-quantification of the VEGF mRNA expression relative to that of β-actin. The values represent the mean ± S.E.M. of at least three separate experiments. Significant differences are indicated by asterisks. **p < 0.01. (TIFF 50 KB)
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Additional file 3: Figure S3: Effects of Cisplatin and Gemcitabine on tumor growth in vivo. Athymic nude mice were inoculated i.p. with A2780CP cells. Two week after inoculation, as described in the Materials and Methods section, the athymic mice were inoculated i.p. with A2780CP cells. (A) Physical appearance of representative mice. The combination treatment with Cisplatin and Gemcitabine reduced tumor production. (B) Magnified views of the tumor in the Vehicle mouse and the histological findings (×200 magnification) of hematoxylin and eosin staining. (C) Relative ratio of the abdominal circumference (I) and tumor weight (II) in each group. The combination therapy with Cisplatin and Gemcitabine significantly decreased the mean abdominal circumference and tumor weight six weeks after the initiation of treatment. Significant differences are indicated by asterisks. **p < 0.01, *p < 0.05. (TIFF 440 KB)
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Additional file 4: Figure S4: Effects of 5-FU on cell viability and Akt phosphorylation in Caov-3. (A) The cells were treated with Cisplatin at various concentrations with (□) or without (×) 100 nM of 5-FU for 24 hours. The number of viable cells was assessed using an MTS assay, as described in the Materials and Methods section. (B) The cells were treated with various combinations of 100 nM of Gemcitabine and 200 μM of Cisplatin for 10 minutes. The cell lysates were subjected to Western blotting for phosphor-Akt (upper panel) and Akt (lower panel), with the density of the control bands arbitrarily set at 1.0. The values represent the mean ± S.E.M. of at least three separate experiments. Significant differences are indicated by asterisks. **p < 0.01. (TIFF 41 KB)
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