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Endocrinology Vol. 147, No. 4 1761-1769
Copyright © 2006 by The Endocrine Society

Inhibition of Phosphatidylinositol 3-Kinase Increases Efficacy of Cisplatin in in Vivo Ovarian Cancer Models

Tsuyoshi Ohta, Masahide Ohmichi, Tadashi Hayasaka, Seiji Mabuchi, Maki Saitoh, Jun Kawagoe, Kazuhiro Takahashi, Hideki Igarashi, Botao Du, Masakazu Doshida, Ishida Gabriela Mirei, Teiichi Motoyama, Keiichi Tasaka and Hirohisa Kurachi

Departments of Obstetrics and Gynecology (T.O., M.O., T.H., M.S., J.K., K.Tak., H.I., B.D., M.D., I.G.M., H.K.) and Pathology (T.M.), Yamagata University, School of Medicine, Yamagata 990-9585 Japan; and Department of Obstetrics and Gynecology (M.O., S.M., K.Tas.), Osaka University Medical School, Osaka 565-0871, Japan

Address all correspondence and requests for reprints to: Dr. Masahide Ohmichi, Department of Obstetrics and Gynecology, Yamagata University, School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan. E-mail: masa{at}med.id.yamagata-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The phosphatidylinositol 3-kinase (PI3K)/Akt cascade has an important role in the resistance of ovarian cancer cells to cisplatin in vitro; however, there have been no reports about whether blocking the PI3K/Akt cascade enhances the sensitivity to cisplatin in vivo. We investigated whether inhibition of PI3K increased the efficacy of cisplatin in an in vivo ovarian cancer model. Blocking the PI3K/Akt cascade with a PI3K inhibitor (wortmannin) increased the efficacy of cisplatin-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated ip with the Caov-3 human ovarian cancer cell line. In addition, wortmannin increased the efficacy of cisplatin-induced apoptosis in tumors cells. There were no detectable side effects in mice treated with wortmannin. Moreover, the antitumor effect of cisplatin detected in mice inoculated with Caov-3 cells stably transfected with empty vector was significantly attenuated, compared with mice inoculated with Caov-3 cells stably transfected with a dominant-negative Akt, K179M-Akt. We confirmed that wortmannin blocked Akt phosphorylation and the downstream targets of the PI3K/Akt cascade, such as BAD (Bcl-2-associated death protein) and nuclear factor-{kappa}B in vivo by immunohistochemical staining and Western blotting. In accordance with the previously reported in vitro results, these in vivo results support the idea that combination therapy with cisplatin and a PI3K inhibitor would increase the therapeutic efficacy of cisplatin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN CANCER is the major cause of death from gynecological malignancy. The current standard therapy for patients with advanced ovarian cancer is cytoreductive surgery followed by the administration of systematic chemotherapy. First-line chemotherapy consists of platinum (cisplatin or carboplatin) in combination with paclitaxel. There was some improvement in survival time with the introduction of platinum and paclitaxel therapy; nevertheless, the likelihood of success in the treatment of women with advanced, recurrent, or persistent ovarian cancer 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, 3, 4, 5, 6, 7, 8, 9). However, the response rates to second-line therapy are strikingly different, depending on the platinum sensitivity of the cancer. It was reported that the interval off platinum-based therapy was a strong predictor of platinum sensitivity (10). Thus, the important determinant of prognosis seems to be whether recurrent ovarian cancer is sensitive or resistant to platinum rather than paclitaxel. Because the prognosis of patients with relapsed ovarian cancer is poor, it is very important to understand how cancer becomes platinum-refractory and to develop molecular targeting therapies for platinum-refractory ovarian cancer.

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 the sensitivity to chemotherapeutic drugs. Akt may promote cell survival by phosphorylating and inactivating the proapoptotic proteins BAD (Bcl-2-associated death protein) and caspase-9 (11, 12). Akt also phosphorylates and inactivates the Forkhead transcription factors, resulting in reduced expression of the cell cycle inhibitor p27Kip1 and the Fas ligand (13, 14, 15). Phosphorylation of inhibitory {kappa}B (I{kappa}B) kinase by Akt also activates nuclear factor I{kappa}B (NF{kappa}B), a transcription factor that has been implicated in cell survival (16, 17). We have previously reported that Akt inactivation sensitizes human ovarian cancer cells to cisplatin (18) and paclitaxel (19). Moreover, the inactivation of a downstream targets of the PI3K/Akt pathway, such as BAD (18) and the transcription factors of Forkhead (20) and NF{kappa}B (21), also sensitize human ovarian cancer cells to cisplatin in vitro. It was previously reported that Akt inactivation by a PI3K inhibitor enhances the sensitivity of cancer to paclitaxel in vivo (22); however, there have been no reports about whether Akt inactivation enhances the sensitivity to cisplatin in vivo.

These considerations led us to examine whether a PI3K inhibitor enhances the sensitivity of ovarian cancer to cisplatin in vivo. In the present study, we show that wortmannin, a known pharmacological inhibitor of PI3K, attenuated the PI3K/Akt cascade, and increased the efficacy of cisplatin in an in vivo ovarian cancer model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Wortmannin was purchased from Cell Signaling (Beverly, MA). The antiphospho-Akt and anti-Akt antibodies, phospho-Akt blocking peptide, and antiphospho-I{kappa}B{alpha} antibodies [immunohistochemistry (IHC) specific] were obtained from Cell Signaling. The phospho Ser136-specific anti-BAD antibody (IHC specific) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The terminal deoxynucleotidyltransferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) kit (ApopTag) was obtained from Chemicon (Temecula, CA).

Cell cultures
The human ovarian mucinous adenocarcinoma cell line Caov-3 was obtained from American Type Culture Collection (Manassas, VA). The cells were cultured at 37 C in DMEM with 10% fetal bovine serum in a water-saturated atmosphere of 95% air-5% CO2.

Constructs
The vector encoding the hemagglutinin-tagged forms of kinase-dead Akt (K179M-Akt) used in this study has been described previously (11, 18).

Treatments in vivo
All of the procedures involving animals in this study were approved by the animal care committee of Yamagata University in accordance with institutional and Japanese government guidelines for animal experiments. Caov-3 cells were harvested in 0.25% trypsin-PBS-EDTA, washed once each with medium and PBS, and resuspended in PBS at 106 cells per 200 µl. One million Caov-3 cells were injected ip into 5-wk-old female nu/nu athymic mice (n = 20). Two weeks after inoculation, one group of mice (n = 5) was treated ip with wortmannin (2 mg/kg) three times weekly plus cisplatin (5 mg/kg) once a week for 4 wk. A second group of mice (n = 5) was treated ip with wortmannin alone (2 mg/kg) three times weekly for 4 wk. A third group (n = 5) was treated ip with cisplatin alone (5 mg/kg) once a week for 4 wk. The remaining mice (n = 5) received vehicle (PBS) alone. Abdominal circumference and body weight were measured twice weekly. At the end of the experiment, mice were killed with CO2. The volume of ascites was measured, and tumor tissue was excised and fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections (5 µm) were used for histochemical analysis.

Immunohistochemical staining
Paraffin-embedded tissue sections were stained with phospho Ser473-specific anti-Akt antibody (IHC specific) and/or phospho-Akt (Ser473) blocking peptide (IHC specific), phospho Ser134-specific anti-BAD antibody (IHC specific), or phospho-specific anti-I{kappa}B{alpha} antibody. Immunohistochemically stained slides were interpreted blindly and independently by a pathologist. Control samples exposed to secondary antibody alone showed no nonspecific staining. Apoptosis was assessed by TUNEL staining using an ApopTag Plus peroxidase in situ apoptosis kit according to the manufacturer’s protocol. Background reactivity for TUNEL was determined by processing slides in the absence of terminal deoxynucleotidyltransferase (negative control). To determine TUNEL expression in tissue sections, we counted the number of apoptotic events in five random fields at x400 magnification and divided that number by the total number of cells per field.

Western blotting
Briefly, tumors were homogenized in 500 µl of lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 100 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride]. Homogenates were centrifuged at 15,000 x g at 4 C for 15 min, and the protein concentrations of the supernatants were determined using a protein assay reagent (Bio-Rad Laboratories (Hercules, CA). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocking was done in 10% BSA in 1x Tris-buffered saline.

Statistics
Statistical analysis was performed using one-way ANOVA followed by Fisher’s least significant difference test, and P < 0.05 was considered significant. Data are expressed as the mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetics of in vivo treatment with wortmannin
To optimize the response to wortmannin, dose-response studies were conducted. Athymic nude mice were inoculated ip with Caov-3 cells (106 cells). Two weeks after inoculation, the mice were randomly placed into four groups that received the following treatments for 4 wk: 1) vehicle control (PBS); 2) wortmannin (0.2 mg/kg) three times a week; 3) wortmannin (2 mg/kg) three times a week; and 4) wortmannin (4 mg/kg) three times a week. Abdominal circumference (Fig. 1AGo) and the volume of ascites at autopsy (Fig. 1BGo) were measured. Treatment with 0.2, 2, and 4 mg/kg wortmannin resulted in a 0.95-, 0.56-, and 0.56-fold decrease in the volume of ascites, compared with the vehicle control, respectively. Treatment with 2 mg/kg wortmannin significantly decreased the abdominal circumference and the volume of ascites, and a similar effect was seen at 4 mg/kg. We therefore used the dose of 2 mg/kg in the following studies.


Figure 1
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  		FIG. 1. Kinetics of in vivo treatment with wortmannin. A and B, Athymic nude mice were inoculated (ip) with Caov-3 cells. Two weeks after inoculation, the mice were randomly placed into four groups that were treated as follows for 4 wk: 1) vehicle control (PBS); 2) wortmannin (0.2 mg/kg) three times a week; 3) wortmannin (2 mg/kg) three times a week; and 4) wortmannin (4 mg/kg) three times a week. Abdominal circumference (A) and the volume of ascites (B) were assessed at the end of the experiment (4 wk of treatment) in the four groups of mice. Values shown represent the mean ± SE. Significant differences are indicated by asterisks. **, P < 0.01, n = 5 in each group. C, Athymic nude mice without injection of ovarian cancer cells were randomly placed into either a vehicle control group (PBS) or a group treated with wortmannin (2 mg/kg) three times a week for 4 wk. Body weight was measured throughout the 28 d of treatment. Values shown represent the mean ± SE. Significant differences are indicated by asterisks. **, P < 0.01, n = 5 in each group.

 
We next examined the toxic effects of 2 mg/kg wortmannin on athymic nude mice without injection of ovarian cancer cells. The mice were randomly placed into two groups that received the following treatments for 4 wk: vehicle control (PBS) and wortmannin (2 mg/kg) three times a week. Body weight was measured (Fig. 1CGo). There were no detectable differences in body weight between the vehicle control group and the wortmannin-treated group, and wortmannin did not cause obvious toxicity.

Effect of wortmannin on the cisplatin-induced inhibition of intraabdominal dissemination of ovarian cancer
We examined the effects of cisplatin or wortmannin alone and in combination on the intraabdominal dissemination of ovarian cancer and ascite formation to assess whether combination therapy would increase the therapeutic efficacy of each agent. Athymic nude mice were inoculated ip with Caov-3 cells. Two weeks after inoculation, the mice were randomly placed into four groups that received the following treatment for 4 wk: 1) vehicle control (PBS), 2) cisplatin (5 mg/kg) once a week, 3) wortmannin (2 mg/kg) three times a week, and 4) the combination of cisplatin (5 mg/kg) once a week + wortmannin (2 mg/kg) three times a week. The appearance of the mice is shown in Fig. 2AGo. Abdominal circumference was measured at the end of 4 wk (Fig. 2BGo). Treatment with cisplatin alone and wortmannin alone resulted in a 4.5 and 11.9% decrease in the abdominal circumference, compared with vehicle control, respectively. The combination of cisplatin + wortmannin resulted in a further decrease in the abdominal circumference (19% reduction). Ascites was observed transperitoneally (Fig. 3AGo) and the volume of ascites was measured at autopsy. Treatment with cisplatin alone and wortmannin alone resulted in a 16 and 44% decrease in the volume of ascites, compared with the vehicle control, respectively. The combination of cisplatin + wortmannin further diminished the production of ascites (88% reduction). Wortmannin also worked synergistically with cisplatin in established tumors (data not shown).


Figure 2
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  		FIG. 2. Appearance and abdominal circumference of mice after treatment with cisplatin, wortmannin, or the combination of both. Athymic nude mice were inoculated (ip) with Caov-3 cells. Two weeks after inoculation, the mice were randomly placed into four groups that were treated as follows for 4 wk: 1) vehicle control (PBS); 2) cisplatin (5 mg/kg) once a week; 3) wortmannin (2 mg/kg) three times a week; or 4) cisplatin (5 mg/kg) once a week + wortmannin (2 mg/kg) three times a week. A, The representative appearance of the mice is shown. B, Abdominal circumference was assessed at the end of the experiment (4 wk of treatment) in the four groups of mice. Values shown represent the mean ± SE. Significant differences are indicated by asterisks. **, P < 0.01, n = 5 in each group. CDDP, Cisplatin.

 

Figure 3
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  		FIG. 3. Ascite formation in mice after treatment with cisplatin, wortmannin, or the combination of both. Athymic nude mice inoculated (ip) with Caov-3 cells were randomly placed into four groups that were treated as described in Fig. 2Go. A, The representative appearance of the mice is shown with the abdominal skin flayed open. B, The volume of ascites was assessed at the end of the experiment (4 wk of treatment) in the four groups of mice described in Fig. 2Go. Values shown represent the mean ± SE. Significant differences are indicated by asterisks. **, P < 0.01; *, P < 0.05; n = 5 in each group. CDDP, Cisplatin.

 
Pathological examinations were done to determine the extent of intraabdominal dissemination of the cancer cells at autopsy. The extent of intraabdominal dissemination is shown visually in Fig. 4AGo. Intraabdominal dissemination was clearly detected in athymic nude mice inoculated ip with Caov-3 cells followed by treatment with vehicle control, and the intraabdominal dissemination was confirmed by the histological findings of mucinous cystoadenocarcinoma (Fig. 4BGo), which was consistent with Caov-3 cells (19).


Figure 4
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  		FIG. 4. Wortmannin enhances the cisplatin-induced inhibition of intraabdominal dissemination. Athymic nude mice inoculated (ip) with Caov-3 cells were randomly placed into four groups that were treated as described in Fig. 2Go. A, At autopsy, pathological examination was performed to determine the extent of intraabdominal dissemination. Both the arrow and arrowhead indicate the intraabdominal dissemination. B, The tumor that had disseminated in the abdomen was fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded tissue sections were stained with hematoxylin and eosin. Scale bars, 15 µm. C, The disseminated tumor was removed and its weight was measured. Values shown represent the mean ± SE. Significant differences are indicated by asterisks. **, P < 0.01; *, P < 0.05; n = 5 in each group. CDDP, Cisplatin.

 
The tumor that had disseminated within the abdomen was measured by weight (Fig. 4CGo). Treatment with cisplatin alone and wortmannin alone resulted in 18 and 39% reduction in the extent of intraabdominally disseminated tumor, compared with vehicle control, respectively. The combination of cisplatin + wortmannin further attenuated the intraabdominal dissemination (81% reduction). These results suggest that combination therapy of cisplatin with wortmannin would increase the therapeutic efficacy of cisplatin.

Confirmation of PI3K as the drug target in vivo
We then tested whether wortmannin blocked the PI3K/Akt cascade in vivo. Tumors that had disseminated intraabdominally were harvested from the mice treated with vehicle control (Fig. 5Go, vehicle), cisplatin alone (Fig. 5Go, CDDP), wortmannin alone (Fig. 5Go, wortmannin), or cisplatin + wortmannin (Fig. 5Go, combination) and were assessed for Akt phosphorylation status by IHC with antiphospho-Akt antibody (Fig. 5Go, left panel). The specificity of the antibody for phosphorylated Akt was confirmed by the finding that the immunoreactivity for phosphorylated Akt was completely inhibited by addition of the phospho-Akt blocking peptide (Fig. 5Go, right panel). Treatment with cisplatin resulted in 83% increase in immunoreactivity for phosphorylated Akt (Fig. 5Go, CDDP), compared with that of vehicle alone (Fig. 5Go, vehicle), as we reported previously by in in vitro studies (18, 23). Immunoreactivity for phosphorylated Akt was completely absent when phospho-Akt blocking peptide was included as a negative control (Fig. 5Go, right panel). Immunoreactivity for phosphorylated Akt in tumors treated with wortmannin alone (Fig. 5Go, wortmannin) and cisplatin + wortmannin (Fig. 5Go, combination) resulted in 61 and 60% decrease by comparison with immunoreactivity for phosphorylated Akt in tumors treated with vehicle alone (Fig. 5Go, vehicle) and cisplatin (Fig. 5Go, CDDP), respectively. These results suggest that wortmannin inhibited both basal and cisplatin-induced Akt phosphorylation in vivo.


Figure 5
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  		FIG. 5. Wortmannin blocks the PI3K/Akt cascade in tumors growing intraabdominally in athymic mice. A, Athymic nude mice inoculated (ip) with Caov-3 cells were randomly placed into four groups that were treated as described in Fig. 2Go. Mice were killed after 4 wk, and tumor tissue was excised and fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded tissue sections were stained with phospho-Akt antibody (left panel) or phosphor-Akt blocking peptide (right panel). Scale bars, 15 µm. B, Phosphorylated Akt cells were expressed as a percentage of total cells. Values shown represent the mean ± SE from evaluation of five random areas at x400 magnification. Significant differences are indicated by asterisks. **, P < 0.01. CDDP, Cisplatin.

 
We further tested whether wortmannin blocked the PI3K/Akt cascade in vivo using tissue homogenate samples. Intraabdominally disseminated tumors harvested from the groups treated with vehicle control, cisplatin alone, wortmannin alone, or cisplatin + wortmannin were homogenized and subjected to Western blotting with antiphospho Akt or anti-Akt antibody. Systemic treatment with wortmannin had no effect on the expression of Akt (Fig. 6Go, bottom panel); however, it significantly inhibited the level of both basal and cisplatin-induced Akt phosphorylation as seen by comparing the vehicle and wortmannin-treated groups or the CDDP and combination groups (Fig. 6Go, middle and top panels). Treated with wortmannin alone (Fig. 6Go, wortmannin) and cisplatin + wortmannin (Fig. 6Go, combination) resulted in 45 and 42% decrease by comparison with Western blotting intensity for phosphorylated Akt in tumors treated with vehicle alone (Fig. 6Go, vehicle) and cisplatin (Fig. 6Go, CDDP), respectively. These results confirmed that drug targeting of the PI3K/Akt cascade occurred in vivo.


Figure 6
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  		FIG. 6. Analysis of the levels of total and phosphorylated Akt protein. Athymic nude mice inoculated (ip) with Caov-3 cells were randomly placed into four groups that were treated as described in Fig. 2Go. Intraabdominal tumors were removed and homogenized. Lysates (250 µg protein) were subjected to Western blotting using antiphospho-Akt (middle panel) or anti-Akt (bottom panel) antibody. The positions of molecular weight markers are noted on the left. Relative densitometric units of the phospho-Akt bands are shown in the top panel, with the density of the vehicle bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01. CDDP, Cisplatin.

 
To rule out the possibility that the effect by wortmannin was due to the systemic effects of inhibition of PI3K, we examined the effect of wortmannin on the levels of phosphorylated Akt in muscle and liver. Although wortmannin reduced the levels of phosphorylated Akt in disseminated tumor (Figs. 5Go and 6Go), it had no effect on the levels of phosphorylated Akt in muscle and liver (data not shown), suggesting that the effect of wortmannin was due to PI3K inhibition in the tumor and not due to systemic effects of inhibition of PI3K.

There still is a possibility that wortmannin is a biologically unstable molecule and is not a very specific PI3K inhibitor. We then compared the effect of cisplatin treatment on mice inoculated with Caov-3 cells stably transfected with empty vector vs. a construct that encodes a dominant-negative Akt, K179M-Akt. The appearance of the mice is shown in Fig. 7AGo. The volume of ascites was measured at autopsy (Fig. 7BGo). The degree of the decrement of the volume of ascites caused by cisplatin in mice inoculated with Caov-3 cells stably transfected with K179M-Akt was more marked than that in mice inoculated with Caov-3 cells stably transfected with empty vector. These results indicate that inhibition of the PI3K cascade further sensitizes the tumor cells to cisplatin in an in vivo ovarian cancer model.


Figure 7
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  		FIG. 7. Transfection with K179M-Akt enhances the cisplatin-induced antitumor effect. Athymic nude mice were inoculated (ip) with or without Caov-3 cells stably transfected with empty vector or K179M-Akt (dominant negative Akt). Two weeks after inoculation, the mice were treated with vehicle control (left panel) or cisplatin (5 mg/kg) once a week by ip injection. A, Representative mice are shown. B, The volumes of ascites were assessed. Values shown represent the mean ± SE. Significant differences are indicated by asterisks. **, P < 0.01; n = 5 in each group. CDDP, Cisplatin.

 
Assessment of apoptosis in intraabdominally dissemination of ovarian cancer
Intraabdominally disseminated tumors harvested from the different groups were processed for the TUNEL staining. The fraction of TUNEL-positive cells was higher in tumors from mice treated with cisplatin + wortmannin than in tumors from mice treated with vehicle alone (Fig. 8AGo). Apoptosis was quantitated in tumors of mice treated with vehicle control, cisplatin alone, wortmannin alone, or cisplatin + wortmannin (Fig. 8BGo). Treatment with cisplatin alone and wortmannin alone resulted in 18- and 11-fold increase in the proportion of apoptotic cells in tumors, compared with vehicle alone. The combination of cisplatin + wortmannin further enhanced the proportion of apoptotic cells in tumors, compared with either agent alone, and resulted in 38-fold increases in the proportion of apoptotic cells in tumors, compared with vehicle alone.


Figure 8
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  		FIG. 8. Analysis of apoptosis in tumors growing intraabdominally in athymic nude mice. Athymic nude mice inoculated (ip) with Caov-3 cells were randomly placed into four groups that were treated as described in Fig. 2Go. A, At autopsy, intraabdominally grown tumors were excised and stained by IHC for TUNEL. Representative areas are shown (x400 magnification). Scale bars, 15 µm. B, TUNEL-positive cells were expressed as a percentage of total cells. Values shown represent the mean ± SE from evaluation of five random areas at x400 magnification. Significant differences are indicated by asterisks. **, P < 0.01. CDDP, Cisplatin.

 
Assessment of the phosphorylation of BAD and NF{kappa}B in intraabdominal dissemination of ovarian cancer
To examine whether wortmannin also blocked the phosphorylation of BAD and NF{kappa}B cascade in vivo, intraabdominally disseminated tumors harvested from the different groups were processed for immunohistochemical analyses with antiphospho-BAD (Fig. 9Go, left panel) or antiphospho-I{kappa}B{alpha} (Fig. 9Go, right panel) antibodies. Treatment with cisplatin resulted in 190% increase in the phosphorylation of BAD and 125% increase in the phosphorylation of I{kappa}B{alpha}, compared with that of vehicle alone, respectively (Fig. 9Go), as we reported previously in in vitro studies (18, 19). Immunoreactivity for phosphorylated BAD in tumors treated with wortmannin alone and cisplatin + wortmannin resulted in 68 and 65% decrease by comparison with immunoreactivity for phosphorylated BAD in tumors treated with vehicle alone and cisplatin alone, respectively (Fig. 9Go, left bottom panel). Immunoreactivity for phosphorylated I{kappa}B{alpha} in tumors treated with wortmannin alone and cisplatin + wortmannin also resulted in 54 and 57% decrease by comparison with immunoreactivity for phosphorylated I{kappa}B{alpha} in tumors treated with vehicle alone and cisplatin alone, respectively (Fig. 9Go, right bottom panel). These results suggest that wortmannin inhibited both basal and cisplatin-induced BAD and I{kappa}B{alpha} phosphorylation in vivo.


Figure 9
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  		FIG. 9. Wortmannin blocks the phosphorylation of BAD and I{kappa}B{alpha} in tumors growing intraabdominally in athymic nude mice. A, Athymic nude mice inoculated (ip) with Caov-3 cells were randomly placed into four groups that were treated as described in Fig. 2Go. At autopsy, intraabdominally grown tumors were excised, and BAD phosphorylation and I{kappa}B{alpha} phosphorylation status were assessed by IHC with antiphospho-BAD antibody (left panel) and antiphospho-I{kappa}B{alpha} antibody (right panel), respectively. Scale bars, 15 µm. B, Phosphorylated BAD (left panel) or phosphorylated I{kappa}B{alpha} (right panel) cells were expressed as a percentage of total cells. Values shown represent the mean ± SE from evaluation of five random areas at x400 magnification. Significant differences are indicated by asterisks. **, P < 0.01. CDDP, Cisplatin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The PI3K/Akt cascade is known to be an important survival factor in the signal transduction cascades involved in the cell growth. This cascade is thus an exciting new target for molecular targeting therapy for cancer. Because the prognosis for patients with relapsed ovarian cancer depends on the sensitivity of patients’ tumors to platinum, it is very important to understand how tumors become refractory to platinum to develop molecular targeting therapies for platinum-refractory ovarian cancer. We previously reported that Akt inactivation sensitizes human ovarian cancer cells to cisplatin (18) in vitro. However, there have been no reports about the effect of Akt inactivation on the sensitivity of cisplatin in in vivo ovarian cancer models. In this study, we used wortmannin to block the PI3K/Akt cascade in vivo ovarian cancer models. We confirmed that wortmannin blocked the Akt phosphorylation (Figs. 5Go and 6Go) and the downstream targets of the PI3K/Akt cascade, such as BAD (Fig. 9Go, left panel) and NF{kappa}B (Fig. 9Go, right panel) in vivo as shown by immunohistochemical staining. We have shown that treatment of athymic mice with the combination of cisplatin + wortmannin caused a more marked reduction of abdominal circumference (Fig. 2Go) and inhibition of ascite production (Fig. 3Go) than treatment with either agent alone. The combination of cisplatin + wortmannin inhibited tumor implantation into the liver and peritoneum more effectively than either agent alone (Fig. 4Go). In addition, the combination of cisplatin + wortmannin further enhanced the increased in the proportion of apoptotic cells than either agent alone (Fig. 8Go). These results suggest that inhibition of the PI3K/Akt cascade enhanced the ability of cisplatin to inhibit the tumor growth and the cisplatin-induced apoptosis in vivo.

How did wortmannin enhance the cisplatin-induced apoptosis (Fig. 8Go) and the ability of cisplatin to inhibit the tumor growth (Figs. 2–5GoGoGoGo)? Cisplatin-induced DNA damage activates PI3K/Akt cascade both in vitro (18) and in vivo, which has been shown to mediate cell survival via the regulation of numerous proteins. Because the balance between cellular survival and apoptotic signals can determine the sensitivity of cells to chemotherapeutic drug, the blockade of PI3K/Akt cascade by wortmannin might promote cisplatin-induced apoptosis and enhance the sensitivity to cisplatin. In addition, it was reported that DNA repair is an important mechanism of resistance to cisplatin (24) and that a PI3K inhibitor (wortmannin or LY294002) led to a decrease in DNA repair activity (25). Therefore, there is a possibility that the blockade of DNA repair activity by wortmannin might be also involved in the mechanism of enhancement of the sensitivity to cisplatin.

Inhibitors of the PI3K/Akt cascade in combination with cisplatin may provide a more effective strategy than single-agent therapy for improving the patient outcome. LY294002, another potent PI3K inhibitor, resulted in a significant dermatological toxicity to achieve growth inhibition of ovarian cancer by LY294002 alone (26). However, the frequency of LY294002 used in combination with paclitaxel was able to reduce and dermatologic toxicity was not detected (22). Thus, although wortmannin at the dose and frequency used in our present study did not cause obvious toxicity (Fig. 1CGo), there is a possibility that increment of either the dose and frequency of wortmannin might induce toxic effects. We showed that the effect of wortmannin may be due to PI3K inhibition in the tumor and not due to the systemic effects of inhibition of PI3K (data not shown). We also confirmed that inhibition of the PI3K cascade sensitizes the tumor to cisplatin by comparing between mice inoculated with Caov-3 cells stably transfected with K179M-Akt and mice inoculated with Caov-3 cells stably transfected with empty vector (Fig. 7Go). Although it was reported that wortmannin inhibits the in vivo growth of human pancreatic cancer (27, 28) and non-small-cell lung cancer (29), this is the first report showing that wortmannin inhibits the in vivo growth of ovarian cancer.

We have reported that the inactivation of downstream targets of the PI3K/Akt cascade, such as BAD (18), the transcription factors Forkhead (20), and NF{kappa}B (21), also sensitizes human ovarian cancer cells to cisplatin. Using small molecular inhibitors to inactivate molecules that mediate cisplatin resistance might be more convenient for clinical application than the genetic approach (23, 30). There are no small molecular inhibitors of BAD or Forkhead. A NF{kappa}B inhibitor sensitized human ovarian cancer cells to cisplatin (21), but it had no effect on the activation of BAD and Forkhead. Because wortmannin blocked not only the Akt phosphorylation (Fig. 5Go) but also the downstream targets of the PI3K/Akt cascade, such as BAD and NF{kappa}B (Fig. 9Go), wortmannin might be more useful for sensitization to cisplatin than inhibition of each molecule.

Inhibitors of the PI3K/Akt cascade increase the efficacy of both paclitaxel (22) and cisplatin in in vivo ovarian cancer models. Because first-line therapy consists of platinum (cisplatin or carboplatin) in combination with paclitaxel, treatment with inhibitors of the PI3K/Akt cascade might make it possible to decrease the dose of first-line therapy, resulting in better compliance by patients, in addition to the improved efficacy of second-line therapy.

In summary, we conclude that wortmannin increases the efficacy of cisplatin in an in vivo ovarian cancer model. Our findings provide valuable information for the preclinical development of treatment protocols for ovarian cancer that target the PI3K/Akt cell survival cascade.


   Footnotes
 
First Published Online January 5, 2006

Abbreviations: BAD, Bcl-2-associated death protein; I{kappa}B, inhibitory-{kappa}B; IHC, immunohistochemistry; NF{kappa}B, nuclear factor-{kappa}B; PI3K, phosphatidylinositol 3-kinase; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine 5-triphosphate nick end labeling.

Received November 18, 2005.

Accepted for publication December 23, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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