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. 2010 Dec 1;10(11):1137–1146. doi: 10.4161/cbt.10.11.13443

Topotecan as a molecular targeting agent which blocks the Akt and VEGF cascade in platinum-resistant ovarian cancers

Satoshi Tsunetoh 1, Yoshito Terai 1,✉, Hiroshi Sasaki 1, Akiko Tanabe 1, Yoshimichi Tanaka 1, Tatsuharu Sekijima 1, Satoe Fujiwara 1, Hiroshi Kawaguchi 1, Masanori Kanemura 1, Yoshiki Yamashita 1, Masahide Ohmichi 1
PMCID: PMC3047105  PMID: 20935474

Abstract

Objective

Topotecan, a novel topoisomerase-1 inhibitor, is a drug that appears to be effective against platinum-resistant ovarian cancers. However, the molecular mechanisms by which Topotecan treatment inhibits cancer cell proliferation are unclear. We investigated whether Topotecan increases the efficacy of Cisplatin in platinum-resistant ovarian cancer models in vitro and in vivo.

Results

Topotecan significantly inhibited Cisplatin-induced Akt activation in Caov-3 cells, but not in A2780 cells. In the presence of Topotecan, Cisplatin-induced growth inhibition and apoptosis were significantly enhanced in Caov-3 cells. Topotecan inhibited not only Cisplatin-induced Akt activation but also VEGF and HIF-1α expression. Moreover, treatment with Topotecan increased the efficacy of Cisplatin-induced growth inhibition in the intraabdominal dissemination and production of ascites in athymic nude mice inoculated with Caov-3 cells.

Methods

We used Cisplatin-resistant Caov-3 cells and Cisplatin-sensitive A2780 cells. We examined the effect of Cisplatin and Topotecan on the cell viability of Caov-3 and A2780 cells by MTS assay. We examined the Akt kinase activity, VEGF and HIF-1α expression after Cisplatin and Topotecan by a western blot analysis. Moreover, we also evaluated the effects of Cisplatin and Topotecan on the intraabdominal dissemination of ovarian cancer in vivo.

Conclusion

We herein demonstrated that Topotecan inhibits Akt kinase activity and VEGF transcriptional activation after Cisplatin treatment in platinum-resistant ovarian cancers. We clarified how Topotecan enhanced the clinical activity in the platinum-resistant ovarian cancer. These results provide a rationale for using Topotecan in clinical regimens aimed at molecular targeting agents in platinum-resistant ovarian cancers.

Key words: topoisomerase-1 inhibitor, ovarian cancer, platinum-resistant, PI3K/Akt cascade, apoptosis, VEGF, topotecan

Introduction

Ovarian cancer is a major cause of death among gynecological malignancies. There has been some improvement in the survival time since the introduction of platinum (Cisplatin or Carboplatin) and Paclitaxel therapy. However, the success rate of treating women with advanced, recurrent, or persistent ovarian cancers has remained largely unchanged for four decades.1 Therefore, there is a need to consider the use of second-line chemotherapeutic options for this cancer.2–8 However, the patient response rates to second-line therapy are strikingly different depending on the platinum sensitivity of the cancer. On the other hand, clear cell carcinoma and mucinous adenocarcinoma in their advanced stages (III and IV) have been reported to show a lower survival rate due to resistance to platinum-based chemotherapy.9–11 Accordingly, an important determinant of the patient prognosis thus seems to be whether or not these ovarian cancers are sensitive or resistant to platinum.

The balance between cellular survival and apoptosis can determine the sensitivity of cells to chemotherapeutic drug-induced apoptosis. Therefore, it is possible that antiapoptotic signals, such as the phosphatidylinositol 3-kinase (PI3K)-Akt survival cascade, are involved in tumor sensitivity to chemotherapeutic drugs. We have previously reported that Akt inactivation sensitizes human ovarian cancer cells to Cisplatin12,13 and Paclitaxel.14 Therefore, inhibition of antiapoptotic signals, such as those medicated by the Akt pathway, has been proposed as a promising strategy to enhance the efficacy of conventional chemotherapeutic agents.15 Since the PI3/Aktcascade is involved in Cisplatin resistance, inhibition of this cascade using gene transfection was effective in reversing Cisplatin resistance.12 Tumor cells secrete vascular endothelial growth factor (VEGF), which increases the proliferation of endothelial cells leading to tumor angiogenesis and subsequent tumor progression.16 Environmental stresses, such as chemotherapy upregulate HIF-1α and VEGF signaling in tumor cells, thus leading to enhanced tumorigenic and angiogenic potential.17 Among the numerous Akt substrates, the mammalian target of rapamycin (mTOR) has been primarily implicated in the regulation of HIF-1α protein at the translocation level.18,19 Therefore, the inhibition of the VEGF cascade will be more effective for blocking Cisplatin resistance. However, small molecular agents which block the Akt and/or VEGF cascade have not yet been discovered.

Topotec an(10-hydroxy-9-dimethylaminomethyl-(S)-camptothecin), a water-soluble camptothecin analog, is a novel topoisomerase I inhibitor which is active against numerous human tumor cell lines and xenograft tumors. Topotecan has also shown clinical activity in ovarian carcinoma, small cell and non-small cell bronchogenic carcinomas and myeloid leukemia. Recently, Phase II trial results showed that Topotecan is effective in both platinum-sensitive and platinum-resistant ovarian cancers.20–24 Preclinical models have demonstrated that Topotecan can enhance platinum-mediated cytotoxicity through inhibition of DNA repair.17,25 Moreover, it was reported that Topotecan induces apoptosis in human lung cancer cells, in part, by downregulating the PI3K-Akt signaling pathway.26

These considerations led us to examine whether Topotecan inhibits the PI3K/Akt signaling pathway in ovarian cancers. Moreover, we evaluated herein whether Topotecan inhibits HIF-1α protein accumulation by downregulation of the PI3k/Akt-mTOR pathway in Cisplatin-resistant ovarian cancers. In the present study, we show that Topotecan attenuates the PI3K/Akt cascade and increases the efficacy of Cisplatin in the Cisplatinresistant ovarian cancer cell line Caov-3 in vitro and in vivo.

Results

Topotecan specifically enhances the Cisplatin-induced inhibition of cell viability.

The sensitivity of Cisplatin in Caov-3 and A2780 cells was examined using a MTS assay. It was first confirmed that A2780 cells are sensitive and Caov-3 cells are resistant to Cisplatin, as reported previously.12 As shown in Figure 1A, the viability of the Caov-3 cells, but not A2780, cells remained unaffected by increasing concentrations of Cisplatin to over 200 µM. There was a synergistic inhibition of cell viability in Caov-3 cells after the combined treatment with Cisplatin and Topotecan (Fig. 1A and B).

Figure 1.

Figure 1

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Cisplatin sensitivity in Caov-3 and A2780 cells was examined using the MTS assay. We examined the effects of Cisplatin or Topotecan alone and in combination on the cell viability of Caov-3 and A2780 cells. by the MTS assay.

Topotecan treatment decreases Akt kinase activity.

We examined the Akt kinase activity after Cisplatin or Topotecan individually and in combination. We observed that Cisplatin induced Akt phosphorylation in Caov-3 cells, but there was no synergistic effect in A2780 cells. Topotecan had no effect on the levels of Akt phosphorylation. However, combination with Cisplatin and Topotecan significantly inhibited the levels of Cisplatin-induced Akt phosphorylation as shown in Figure 2A. Treatment with Cisplatin and Topotecan resulted in a 67% decrease in comparison to the western blotting band intensities of phosphorylated Akt in Caov-3 cells treated with Cisplatin alone.

Figure 2A–C.

Figure 2A–C

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Topotecan inhibits Akt- and mTOR-phosphorylation induced by Cisplatin in Caov-3 cells. Caov-3 (A, C and D) and A2780 (B and E) cells were treated, as described in Materials and Methods. Cell lysates were subjected to SDS-PAGE, followed by a western blot analysis with an anti-phospho-Akt (Ser473) antibody (top parts of A and B), an anti-Akt antibody (bottom of A and B), an anti-PARP antibody (bottom of C), an anti-phospho-mAkt antibody (top of D) and an anti-mTOR antibody (bottom of D). The respective band densitometry analyses were performed with the Image J software program. The values represent the means ± SE from at least three independent experiments. Significant differences are indicated by asterisks.

**p < 0.01.

We examined whether Topotecan affects Akt activity, which was induced by Cisplatin in Caov-3 cells. PARP is a substrate of caspase-3 and was also cleaved to produce the 85 kDa apoptotic fragment.28 Topotecan significantly induced the cleavage of PARP, but Cisplatin did not induce PARP cleavage in Caov-3 cells (Fig. 2C). These results suggested that Topotecan promotes apoptosis via the suppression of Akt kinase activity, which was induced by Cisplatin, in Caov-3 cells.

Topotecan blocks hypoxia-induced factor-1α and vascular endothelial growth factor expression which are induced by Cisplatin.

High levels of VEGF expression and increased microvessel densities are associated with a poor survival of patients with advanced stage of ovarian cancer.27 A major regulator of VEGF is the hypoxia-inducible factor (HIF)-1α. We observed that Cisplatin induces not only Akt but also mTOR phosphorylation in Caov-3 cells (Fig. 2D and 2nd line); however, there was no such synergistic effect in A2780 cells (data no shown). In addition, Topotecan did not affect the expression of mTOR phosphorylation. However, combined treatment with Cisplatin and Topotecan significantly inhibited the levels of Cisplatin-induced mTOR phosphorylation (Fig. 2D and 4th line). According to the findings of a western blot analysis, treatment with Cisplatin and Topotecan resulted in an 89.2% decrease in phosphorylated mTOR in Caov-3 cells compared to cells treated with Cisplatin alone. Therefore, we speculated that Cisplatin might be affecting VEGF expression through the Akt/mTOR-HIF-1α cascade in Cisplatin-resistant ovarian cancer cells. Accordingly, we examined whether Cisplatin treatment affects VEGF expression in Caov-3 cells. HIF-1α exists in a dimer, comprised of HIF-1α and HIF-1β. Which are the major transcriptional modulators of VEGF. Cisplatin stimulated marked HIF-1α translocation into the nucleus (Fig. 3A middle part, 2nd line), but both total HIF-1α levels (Fig. 3A top) and HIF-1β levels (data not shown) were also affected. Next, we evaluated whether Topotecan blocked HIF-1α translocation into the nucleus as induced by Cisplatin. Topotecan significantly inhibited the ability of Cisplatin to induce the translocation of HIF-1α (Fig. 3A and 4th line), whereas Topotecan alone did not affect the localization of HIF-1α in Caov-3 cells (Fig. 3A and 3rd line). To directly evaluate whether HIF-1α played a role in stimulating VEGF protein expression, we evaluated whether HIF-1α was recruited to the promoter of the VEGF gene by chromatin immunoprecipitation (ChIP) assay, as seen in Figure 3B and C. Caov-3 cells and A2780 cells were treated with Cisplatin and lysates were chromatin-immunoprecipated with an antibody against HIF-1α. The ChIP-captured DNA was subjected to PCR amplification using PCR primers located upstream of the hypoxia response element (HRE)-site of the VEGF promoter.30 Cisplatin induced the binding of HIF-1α to the HRE binding site of the VEGF promoter in Caov-3 cells (Fig. 3B and 2nd line), but not in A2780 cells (Fig. 3C and 2nd line). Topotecan significantly inhibited the ability of Cisplatin to induce the binding of HIF-1α to the HRE binding site of the promoter of VEGF in Caov-3 cells (Fig. 3B and 4th line). These results suggest that HIF-1α. Which is induced by Cisplatin, plays a role in stimulating the VEGF gene in Caov-3 cells, but not in A2780 cells. We examined the VEGF expression in Caov-3 cells treated with vehicle, Cisplatin alone, Topotecan alone, or the combination of Cisplatin and Topotecan, by a real time-PCR analysis (Fig. 3D). The combination of Cisplatin and Topotecan significantly decreased the expression of the VEGF gene compared with Cisplatin alone. These results indicate that combination therapy of Cisplatin and Topotecan would inhibit HIF-1α and VEGF expression which are induced by Cisplatin treatment.

Figure 2D and E.

Figure 2D and E

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Topotecan inhibits Akt-and mTOR-phosphorylation induced by Cisplatin in Caov-3 cells. Caov-3 (A, C and D) and A2780 (B and E) cells were treated, as described in Materials and Methods. Cell lysates were subjected to SDS-PAGE, followed by a western blot analysis with an antiphospho-Akt (Ser473) antibody (top of A and B), an anti-Akt antibody (bottom of A and B), an anti-PARP antibody (bottom of C), an anti-phospho-mAkt antibody (top of D) and an anti-mTOR antibody (bottom of D). The respective band densitometry analyses were performed with the Image J software program. The values represent the means ± SE from at least three independent experiments. Significant differences are indicated by asterisks.

**p < 0.01.

Figure 3.

Figure 3

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The effects of kinase inhibitors on Cisplatin-induced HIF-1α stabilization. (A) The proteins in the nuclear fractions and whole cell lysates were isolated and subjected to a western blot analysis. (B and C) Cell lysates were chromatin immunoprecipitated with antibody against HIF-1α as described in Materials and Methods. (D) Topotecan inhibits Cisplatin-induced VEGF-mRNA expression in Caov-3 cells. The values represent the mean ± SE , n = 3. **p < 0.01.

Effect of topotecan on cisplatin-induced inhibition of intraabdominal dissemination of ovarian cancers.

Peritoneal dissemination is the primary route of progression in human ovarian cancer and the amount of ascites and disseminated tumor burden correlates with patient prognosis in humans.31 We therefore examined the effect of Cisplatin and Topotecan alone and in combination on the control of intraabdominal dissemination of ovarian cancers, ascites formation and tumor growth to assess whether combination therapy would increase the therapeutic efficacy of each agent. Athymic nude mice were inoculated i.p. with Caov-3 cells, as described in Materials and Methods. The appearance of the mice is shown in Figure 4A, I. Intraabdominal dissemination was clearly detected in athymic nude mice inoculated i.p. with Caov-3 cells followed by treatment with PBS (Fig. 4A, II and III). The combination of Cisplatin and Topotecan further enhanced the inhibitory effects on the production of ascites and on intraabdominal dissemination (Fig. 4A, III). After performing a histological examination (Fig. 4B), these abdominal tumors were found to be papillary adenocarcinomas, which is consistent with Caov-3 cells. The mean abdominal circumferences 6 weeks after initiating treatment in the mice treated with combination therapy of Cisplatin and Topotecan were significantly lower than in mice treated with PBS or Cisplatin alone (Fig. 4C), suggesting that ascites production was inhibited by treatment with Topotecan. Surprisingly, no macroscopic tumor implants were detected in mice treated with Cisplatin and Topotecan (Fig. 4A, III).

Figure 4.

Figure 4

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Effects of Cisplatin and Topotecan on tumor growth in vivo. Athymic nude mice were inoculated i.p. with Caov-3 cells. Two week after inoculation, as described in Materials and Methods. (A) Athymic mice were inoculated i.p. with Caov-3 cells. I, II, physical appearances of representative mice; III, magnified views of the intraabdominal dissemination pattern of the same mouse, respectively. (B) Histological findings (x200 magnification) of hematoxylin and eosin staining of parietal peritoneal dissemination of athymic nude mice. Scale bars represent 100 µm.

(C) Abdominal circumferences of groups (cm). Significant differences are indicated by asterisks. **p < 0.01, *p < 0.05. (D) VEGF-ELISA of ascites for groups.

Topotecan inhibits angiogenic activity induced by Cisplatin in the intra-abdominal disseminated ovarian cancer model.

We next examined whether Topotecan decreases the VEGF expression in vivo. Figure 4D shows the concentration of VEGF in ascitic fluids which were present in an intra-abdominal disseminated ovarian cancer in mice. VEGF expression was decreased significantly upon combined treatment with Cisplatin and Topotecan compared to VEGF expression in vehicle, Cisplatin alone or Topotecan-treated mice (Fig. 4D). These results indicate that Cisplatin and Topotecan combination therapy significantly inhibits angiogenic activity.

Discussion

Resistance to Cisplatin is a multifactorial phenomenon, the elements of which may be placed in three general categories: (a) reduced intracellular accumulation of Cisplatin, (b) elevated levels of glutathione and metallothionein and (c) increased DNA damage tolerance or repair.30,32,33 Because Cisplatin acts by forming intrastrand and interstrand DNA cross-links and DNAprotein cross-links, thus resulting in DNA damage, overcoming these lesions by heightened repair is an important mechanism for Cisplatin resistance.34 We have previously described that the PI3K/Akt cascade is involved in Cisplatin resistance.12–14 Although it is well known that Topotecan is the most frequently administered drug in platinum-resistant ovarian carcinoma, the mechanisms underlying these phenomena are not yet characterized. We found that combination treatment with Cisplatin and Topotecan significantly inhibits the level of Cisplatin-induced Akt activity in Caov-3 cells. We clarified that Topotecan exerts its cytotoxic effects by interfering with antiapoptotic machinery and Topotecan significantly enhances PARP cleavage. We found that Cisplatin-induced HIF-1α directly binds the HRE binding site of the VEGF promoter and regulates VEGF expression in Caov-3 cells. The inhibition of VEGF may represent a novel Topotecan mechanism, in which Topotecan induces cellular apoptosis and inhibits tumor angiogenesis in ovarian cancers. Moreover, we found that the combined treatment of Cisplatin and Topotecan significantly inhibits intra-abdominal tumor cell dissemination, ascites production and the concentration of VEGF in ascetic fluid compared to treatment with Cisplatin or Topotecan alone. These results suggested that the cytotoxic effects of Topotecan might be mediated in part by suppressing Akt kinase activity, which is Cisplatin-induced and may cause cellular apoptosis in platinum-resistant ovarian cancers.

A previous clinical study did not examine the response rates to Topotecan with Cisplatin in those patients with platinumresistant ovarian cancers. Irinotecan (CPT-11) which is an agent of topoisomerase I inhibitor and Cisplatin have both been reported to be effective in the treatment of patients with clear cell carcinoma.35 However, only a small number of patients were investigated in the previously reported studies. The response rate to Topotecan and Carboplatin in those patients with recurrent ovarian cancers is approximately 8.7 to 70%.36–38 We were unable to show whether other factors, such as reduced accumulation of Cisplatin or the elevated levels of glutathione and metallothionein, affect the resistance of Cisplatin-resistant ovarian cancer. This additional knowledge might be helpful for future strategies to more effectively circumvent the multifactorial mechanisms of platinum resistance. Topoisomerase I inhibitor (CPT-11) and Cisplatin are currently being evaluated by the Gynecologic Cancer Intergroup/Japanese Gynecologic Oncology Group 3017. This trial is designed to evaluate the efficacy of the response rates to Topoisomerase I inhibitor with Cisplatin in patients with clear cell carcinoma. We believe that our data support the scientific justification for both this and future trials with Topotecan in patients with platinum-resistant ovarian cancers.

In conclusion, we herein demonstrated that Topotecan inhibits Akt kinase activity and VEGF transcriptional activation after Cisplatin treatment in platinum-resistant ovarian cancers. These results provide a rationale for using Topotecan in clinical regimens aimed at molecular targeting agents in platinum-resistant ovarian cancers.

Materials and Methods

Reagents/antibodies.

Topotecan was purchased from Sigma-Aldrich (Dorset, UK) and dissolved in sterile water. Cisplatin was also purchased from Sigma-Aldrich (Dorset, UK). The antiphospho-Akt (ser473), anti-Akt antibodies, anti-phospho PARP, and anti-HIF-1α antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Cell lines.

The human ovarian mucinous adenocarcinoma Caov-3 cell line was obtained from the American Type Culture Collection (Rockville, MD), and the human ovarian cancer A2780 cell line was kindly provided by Dr. Tsuruo (Institute of Molecular and Cellular Biosciences, Tokyo, Japan)27 and RF. Ozols and TC. Hamilton (NCI, National Institutes of Health, Bethesda, MD).14 The cells were cultured at 37°C/5% CO2 in DMEM supplemented with 10% FBS in a humidified atmosphere.

Proliferation assay.

Changes in cell proliferation were examined by the addition of Cisplatin at various concentrations for 48 hours one day after seeding test cells into 96 well plates. The number of surviving Caov-3 and A2780 cells was determined after 24 hours of treatment by measuring the dissolved formazan products after the addition of MTS as described by the manufacturer (Promega). All experiments were carried out in quadruplicate, and the cell viability was expressed as the ratio of the number of viable cells with Cisplatin treatment to those without treatment.

Western blot analysis.

The cells were starved and treated with PBS or 200 µM Cisplatin for 24 hours with or without 1 µM Topotecan for 36 hours. Cells were washed twice with ice-cold phosphate-buffered saline, lysed, and separated to cytoplasmic and nuclear fractions using the Nuclear Extract Kit according to the manufacturer's protocol (Active Motif, Carlsbad CA). To detect Akt, phosphorylated Akt, mTOR, phosphorylated mTOR or PARP proteins, equal amounts of cytoplasmic proteins were separated, and to detect HIF-1α proteins in the nuclear fraction, equal amounts of nuclear proteins were separated by SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes. Nonspecific antigen sites were blocked with 10% bovine serum albumin in 1x Tris-buffered saline. Western blot analyses were performed with various specific primary antibodies.

Chromatin immunoprecipitation (ChIP) assays.

ChIP assays were carried out according to the manufacturer's protocol. Briefly, ovarian cancer cell extracts were sonicated to shear chromatin to an average size of 600 kb. The extracts were divided into aliquots, and anti-HIF-1α antibodies were added to the aliquots at a 1:100 dilution for immunoprecipitation. After immunoprecipitation, an aliquot of each captured immunocomplex was subjected to a western blot analysis to confirm that the captured chromatin contained HIF-1α corresponding to the specificity of the antibody that had been used for ChIP. DNA was purified using a MinElute Reaction Cleanup kit (QIAGEN) and resuspended in 10 µl of 1x TE. The purified ChIP-captured DNA was analyzed by PCR. PCR amplifications were performed with the following specific primer pairs designed for each gene promoter: VEGF, 5′-AAG ACA TCT GGC GGA AAC C-3′ (forward) and 5′-ACA ATT GGT CGC TAA CCG AG-3′ (reverse). The PCR products were separated by electrophoresis on a 2% agarose gel.

Real-time polymerase chain reaction (PCR).

Caov-3 cells were treated with PBS, Cisplatin, Topotecan or Cisplatin plus Topotecan, for 36 hours. Synthesized cDNAs were diluted to a final concentration of 20 ng/µl and 50 ng were used per reaction. PCR primers for the Taqmen/Probe Library assays were designed with the Probe Library Assay Design Center (Roche), and are listed as follows. VEGF 5′-TTG AGT TAA ACG AAC G-3′ (forward) and 5′-GGT TCC CGA ACC CTG AG-3′ (reverse); GAPDH 5′-AGC CAC ATC GCT CAG ACA-3′ (forward) and 5′-GCC CAA TAC ACC AAA TC C-3′ (reverse). Quantitative real-time PCR was performed on a LightCycler 2.0 (Roche Diagnostics, Indianapolis, IN) and results were analyzed by LightCycler Software program 4.05 (Roche Diagnostics) using a calibrator-normalized relative quantification approach. Relative gene expression quantification was normalized to GAPDH expression.

In vivo growth inhibition assay.

Female 6-week-old athymic nude mice (BALB/c Slc-nu/nu) were used for tumor experiments. All mice were purchased from Japan SLC, Int and were housed five mice per cage. The mice had access to sterile food pellets and water ad libitum. The institutional guidelines for animal welfare and experimental conduct were followed. Caov-3 cells were suspended in PBS. Then 5 × 106 cells were injected i.p. into each of the 20 female, 6-week-old nude mice. Two weeks after inoculation, athymic nude mice were inoculated i.p. with Caov-3 cells and randomly assigned into four groups treated with the following for 6 weeks: (a) vehicle (PBS); (b) Cisplatin (5 mg/kg) once a week; (c) Topotecan (12.5 mg/kg) once a week; and (d) Cisplatin (5 mg/kg) once a week + Topotecan (12.5 mg/kg) once a week. The abdominal circumference was measured weekly. Six weeks after initiating treatment, all mice were sacrificed, and the presence of macroscopic disease and the ascites volumes were examined.

Statistics.

The statistical analysis was performed using a oneway ANOVA followed by a Fisher's least significant difference test, and differences with p-values less than 0.05 were considered statistically significant. Data are expressed as the mean ± SE.

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas, No.19591946 (to Y.T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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