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Departments of Cell Biology (J.J.P., X.L., K.P.C., K.P.) and Pathology and Laboratory Medicine (M.M.S.) and the Center for Vascular Biology (K.P.C., K.P.), University of Connecticut Health Center, Farmington, Connecticut 06030
Address all correspondence and requests for reprints to: John J. Peluso, Ph.D., Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: peluso{at}nso2.uchc.edu.
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Abstract |
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Objective: The objective was to determine whether P4 acts through progesterone receptor membrane component-1 (PGRMC1) in ovarian cancers.
Design, Setting and Patients: Archival tissue and cDNA provided by OriGene were used for expression studies. In vitro experiments were conducted with Ovcar-3 cells.
Main Outcome Measures: PCR, Western blot, and immunohistochemistry were used to measure expression of PGRMC1 and nuclear progesterone receptor (PGR). PGRMC1’s role in regulating the viability of ovarian cancers was assessed by overexpressing PGRMC1, depleting PGRMC1 using small interfering RNA, and attenuating PGRMC1’s action with a blocking antibody. Apoptosis was determined by 4',6'-diamino-2-phenylindole staining.
Results: PGRMC1 mRNA increased and PGR mRNA decreased in advanced stages of ovarian cancer. Unlike PGR, PGRMC1 was expressed in virtually every cancer cell within the tumor. A similar relationship between PGRMC1 and PGR was observed in Ovcar-3 cells. In these cells P4 suppressed apoptosis induced by either serum withdrawal or cisplatin (CDDP). Moreover, in the presence of P4, the following occurs: 1) overexpression of PGRMC1 reduces the effectiveness of CDDP, 2) depletion of PGRMC1 with small interfering RNA enhances the effects of CDDP, and 3) PGRMC1 antibody treatment increases the apoptotic response to CDDP.
Conclusions: These findings indicate that PGRMC1 plays an important role in promoting ovarian cancer cell viability and that attenuating PGRMC1’s action makes the ovarian cancer cells more sensitive to CDDP. These data suggest that targeted depletion of PGRMC1 could be useful as an adjunct to CDDP therapy.
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Introduction |
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There are many theories as to why cancer cells are resistant to platinum-based chemotherapy. In the case of ovarian cancer, sex steroids such as progesterone (P4) may play an important role (2, 3, 4, 5). P4 is produced by numerous cell types within the ovary (6) including ovarian surface epithelial cells (7) and the ovarian cancers that are thought to be derived from these epithelial cells (4, 8). In addition P4-containing contraceptives and pregnancy, both of which are associated with high levels of P4, appear to provide a protective effect for ovarian cancers (4). This protective effect may be mediated by high levels of P4 that inhibit proliferation and ultimately promote apoptosis (9, 10, 11).
Although the mechanism has not been conclusively defined, P4 is generally thought to mediate its protective action through its well-characterized nuclear receptor [progesterone receptor (PGR)] because its expression is a positive predictor of patient survival (12, 13, 14). The rationale for the involvement of PGR in P4’s protective action is also supported by the loss of heterozygosity at chromosome 11q23.3–24.3, which is the locus for the PGR (15, 16, 17). This loss of heterozygosity is observed in about 75% of ovarian cancers and associated with a poor prognosis (15, 16, 17).
Although the clinical and genetic observations suggest that P4 might be used to suppress ovarian cancer, the data from limited clinical trials have been very disappointing (4, 18). In addition, more recent studies suggest that there may be an inverse relationship between P4 and ovarian cancer (5). One reason for this disparity may be related to an incomplete understanding of P4’s actions in ovarian cancers, specifically the assumption that all of P4’s actions are mediated through the PGR. This may not be the case because recent studies have shown P4 can regulate the viability of normal ovarian cells by activating a membrane P4 receptor, referred to as progesterone receptor membrane component-1 (PGRMC1) (19, 20). This receptor has also been detected in breast cancers, and it appears to make breast cancer cell lines resistant to chemotherapy (21, 22). Based on these findings, the present studies were designed to: 1) determine whether PGRMC1 is expressed in ovarian cancers 2) compare its expression pattern with that of PGR, and 3) assess its ability to regulate the viability of ovarian cancer cells in vitro.
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Materials and Methods |
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For these studies, ovarian cancer specimens were obtained from the following sources: 1) provided by OriGene (Rockville MD), 2) archival specimens obtained from the Department of Pathology, University of Connecticut Health Center, or 3) an ovarian cancer cell line (Ovcar-3 cells) provided by Dr. Nellie Auersperg (University of British Columbia, Vancouver, Canada).
Ovarian cancer I quantitative PCR tissue arrays were purchased from OriGene and used to assess the expression of PGR and PGRMC1. This array consisted of cDNA obtained from 48 ovarian cancers. All the clinical information associated with each of these samples can be found on the OriGene web site (ovarian cancer panel 1; http://www.origene.com/geneexpression/disease-panels/products/HORT101.aspx).
To determine the localization and number of cells that express PGR and PGRMC1, 20 archival specimens were obtained from the Department of Pathology. These specimens were all serous carcinomas and were stage Ia (n = 2), stage Ic (n = 2), stage IIc (n = 2), stage IIIb (n = 1), stage IIIc (12), and stage V (n = 1) tumors. Sections from these tumors were immunostained for either PGR or PGRMC1. These specimens were obtained with institutional review board approval.
To assess the effects of CDDP’s, P4’s, and PGRMC1’s role in regulating the viability of ovarian cancer cells, Ovcar-3 cells were used. Ovcar-3 cells were maintained in culture as described by Kim et al. (23). Briefly, Ovcar-3 cells were cultured in RPMI 1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 U/ml penicillin G, 100 µg/ml streptomycin (Invitrogen), at 37 C in a humidified atmosphere of 5% CO2 incubator. The Ovcar-3 cells were cultured for 72 h before each experiment.
Expression of PGR and PGRMC1
Several techniques were used to monitor the expression of PGR and PGRMC1. These include real time PCR, RT-PCR, immunohistochemistry, immunocytochemistry, and Western blot analysis.
Real time PCR Quantitative measurements of PGR and PGRMC1 mRNA were made on cDNA samples provided by OriGene. Briefly, primers to human PGR and human PGRMC-1 were designed using ABI Prism Primer Express 2.0 software (Applied Biosystems, Foster City, CA). Primers were evaluated with National Center for Biotechnology Information Blast (Bethesda, MD) to confirm product specificity, and products were designed to cross intron/exon borders. Primers used were: PGR forward, 5'-CATGGTCCTTGGAGGTCGAA-3' and reverse, 5'-GAGAGCAACAGCATCCAG TGC-3' and PGRMC-1 forward, 5'-CAACGGCAAGGTGTTCGAT-3' and reverse, 5'-TCCAGCAAAGACCCCATACG-3'.
Quantitative real time PCR was performed on the MyiQ single-color real-time PCR detection system (Bio-Rad, Hercules, CA). Samples were resuspended in 25 µl of the iQ SyberGreen Supermix (Bio-Rad) containing appropriate primers. Relative gene expression was evaluated with Bio-Rad iQ5 software using the change in cycle threshold value method (24). Each gene was evaluated on two separate identical array plates, which were loaded with equal amounts of cDNA per well as described by the manufacturer. End products were also run on agarose gels to confirm product size.
RT-PCR RT-PCR was used to characterize the expression of PGR and PGRMC1 in Ovcar-3 cells. In this protocol, total RNA was isolated from Ovcar-3 cells using RNeasy Plus minikit (QIAGEN, Valencia, CA). Then cDNA was synthesized by incubating 1 µg of RNA with oligo-dT and Muloney murine leukemia virus reverse transcriptase (Invitrogen). Primers for the subsequent PCRs were as follows: PGR forward, 5'-GTGCAAGGTTGGAGACAGCT and reverse, 3'-TTTGCCCTTCAGAAGCG GAC (213 bp); PGRMC1 forward, 5'-GAGGATGTGGTGGCGACT and reverse, 3'-TAATCATTTTTCCGGGCACT (578 bp). As a positive control for PGR, mRNA isolated from granulosa/luteal cells obtained from patients undergoing in vitro fertilization was used. The protocol involving the use of these cells was approved by the institutional review board as previously published (25).
Immunological detection of PGR and PGRMC1 PGR and PGRMC1 were localized in 5-µm paraffin sections of formalin-fixed ovarian tumors. To block endogenous peroxidase activity, sections were incubated in 0.3% peroxidase in methanol for 30 min at room temperature. To reduce nonspecific staining, the slides were incubated with Powerblock (Biogenex, San Roman, CA).
PGR was detected by incubating the PGR antibody (1:50 dilution Ab-8; Lab Vision/Neomarker, Fremont, CA) overnight. The epitope for PGR antibody is the N-terminal half of the human PGR. It is a mouse monoclonal antibody that detects both the A and B forms of PGR. After incubation with either the PGR antibody or IgG (negative control), the sections were incubated with biotinylated goat antimouse IgG followed by a 30 min incubation with ABC reagent (Vector Laboratories, Burlingame, CA). The slides were then developed using a diaminobenzidine-peroxidase substrate for 5 min followed by light counterstaining with methyl green. The presence of PGR was revealed by the presence of a reddish brown precipitate.
Immunohistochemistry and immunocytochemistry were also used to assess the localization of PGRMC1 in formalin-fixed paraffin-embedded tissue sections of ovarian cancers and Ovcar-3 cells, respectively. The Ovcar-3 cells were plated on coverslips that were placed in 35-mm culture dishes, cultured for 3 d, fixed with 10% formalin, and then incubated with 0.1% Triton X-100. The PGRMC1 antibody used to stain the tissue sections and cells was a rabbit antibody built against 15 N-terminal amino acids of porcine PGRMC1 (26, 27). This antibody was previously used to detect PGRMC1in human granulosa/luteal cells (25). The tissue sections and coverslips were incubated overnight at 4 C with the antibody to PGRMC1 (1:50). After washing to remove the primary antibody, the slides/coverslips were incubated for 1 h at room temperature in the dark with either biotinylated goat antirabbit IgG followed by a 30 min incubation with ABC reagent (Vector Laboratories) or Alexa Fluor 488-goat antirabbit IgG (1:100). Negative controls were also processed as previously described. The slides and coverslips were then observed under bright-field, epifluorescent, or confocal optics, depending on the experimental design.
For Western blot studies, Ovcar-3 cells were lysed in radioimmunoprecipitation assay buffer [50 mM Tris, 150 mM sodium chloride, 1.0 mM EDTA, 1% Nonidet P-40, and 0.25% sodium-deoxycolate (pH 7.0)], which was supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail 1 (Sigma Chemical Co., St. Louis, MO). The lysate was then centrifuged at 1000 x g at 4 C for 5 min. Aliquots of this preparation were run on a 10% acrylamide gel and transferred to nitrocellulose. The nitrocellulose was then incubated with 5% nonfat dry milk overnight at 4 C. The nitrocellulose blot was then incubated with the rabbit PGRMC1 antibody (1:2000) (25) for 1 h at room temperature. Western blots were processed using a horseradish peroxidase goat antirabbit antibody (1: 5000). ECL Western blotting analysis system (Amersham Biosciences, Piscataway, NJ) was used to reveal PGRMC1. As a negative control, rabbit IgG was used in place of the PGRMC1 antibody. Western blots were also conducted using green fluorescent protein (GFP; 1:2000; Cell Signaling Inc., Beverly, MA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (1: 4000; Ambion Inc., Austin, TX) according to the manufacturer’s instructions.
In vitro modulation of PGRMC1 levels in Ovcar-3 cells
Three approaches were used to alter the levels or activity of PGRMC1. They include overexpression, depletion using PGRMC1 small interfering RNA (siRNA), and interference with PGRMC1’s activity using a blocking antibody.
To increase the levels of PGRMC1, Ovcar-3 cells were transfected with a GFP-PGRMC1 expression vector using a lipofectamine-based protocol as previously described (28). This construct was made by cloning the entire coding region of human PGRMC1 into the pEGFP-N1 vector. This construct was provided by Drs. Wehling and Losel of the University of Heidelberg.
PGRMC1 siRNA treatment was used to deplete PGRMC1 levels (28). In this protocol, PGRMC1 depletion was performed by transfecting PGRMC1 siRNA using siPORT NeoFX transfection protocol outlined by Ambion. Studies were conducted in which either scramble (control) siRNA (catalog no. AM4611) or one of three predesigned human PGRMC1 siRNAs (Ambion siRNA ID no. 18430, 18340,18248) were transfected at a concentration of 30 nM. Levels of PGRMC1 mRNA and protein were assessed by real-time PCR and Western blot, respectively, as previously described. In addition, immunocytochemical analysis confirmed that PGRMC1 siRNA (ID no. 18248) was an effective PGRMC1 siRNA. This siRNA targeted exon 1 of PGRMC1 (forward, 5'-GGUGUUCGAUGUGACCAAAtt and reverse, 5'-UUUGGUCACAUCGAACACCtt). Based on this, Ovcar-3 cells were cultured for 3 d and then treated for 3 d with either scramble or PGRMC1 siRNA (ID no. 18248). The cells were then treated for 24 h with CDDP (15 µM) and P4 (1 µM) and observed apoptosis as described below.
Finally, a blocking antibody study was conducted as previously described (25). Ovcar-3 cells were plated and cultured for 3 d as previously described. The cells were then washed and cultured for 24 h with serum supplemented containing CDDP (15 µM) and P4 (1 µM) and either rabbit IgG (20 µg/ml) medium or the antibody to PGRMC1 (20 µg/ml). After culture, the cells were rinsed in Krebs/HEPES buffer and stained to detect apoptotic nuclei as described below.
Detection of apoptotic nuclei
Regardless of the experimental design, in situ DNA staining was used to identify apoptotic nuclei by adding 4',6'-diamino-2-phenylindole (DAPI) directly to the culture medium at a final concentration of 2 µg/ml. The cultures were incubated for 10 min at 37 C in the dark. After staining, the cells were observed under epifluorescent optics. Under these conditions only cells with condensed or fragmented nuclei were stained intensely with DAPI. The DAPI-stained cells also stained with YOPRO-1, a well-known nuclear stain that identifies apoptotic cells (19, 25, 28). These cells were considered to be apoptotic (25). At least 100 cells/culture well were counted and the percentage of apoptotic nuclei in each well determined. [See the data shown in Figs. 5
and 8; cells were selected randomly. Also see the data in Fig. 6 in which the apoptotic status (i.e. DAPI staining; blue fluorescence) was determined for 100 transfected cells per well as indicated by their GFP (i.e. green) fluorescence.]
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All experiments were repeated two to three times. The apoptosis studies were usually conducted in triplicate. When appropriate, the data were pooled to generate means ± SEs and analyzed by either a Student t test when an experiment consisted of two treatment groups or a one-way ANOVA followed by a Student-Newman-Keuls test if more than two treatments groups were being compared. Due to heterogeneity of variance, the PGRMC1/PGR mRNA data were log transformed before analysis. Regardless of the statistical test used, P < 0.05 was considered to be significant.
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Results |
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). PGRMC1 mRNA was also readily detected by real-time PCR in normal ovarian tissue (Fig. 1B), with its level of expression being 2- to 4-fold greater than that of PGR (Fig. 1C). PGRMC1 mRNA levels remained relatively constant in stages I and II ovarian cancers and then increased in stage III-IV ovarian cancers (P < 0.05; Fig. 1B). As might be predicted, the ratio of PGRMC1 to PGR mRNA increased with advancing stage of ovarian cancer (P < 0.05; Fig. 1C). Moreover, when all samples were analyzed, there was an inverse relationship between the PGRMC1 mRNA to PGR mRNA ratio and PGR mRNA levels (Fig. 1D; r = 0.76).
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, PGR was expressed by some but not all cells of an ovarian cancer. Moreover, PGR was not detected in all areas of PGR-positive tumors. Conversely, PGRMC1 was detected in virtually all of the cells of ovarian tumors (Fig. 2C), even in cells that do not express PGR (compare cells marked with arrows in Fig. 2, A and C). In most ovarian cancers, PGR was not detected (Fig. 2B), whereas PGRMC1 was highly expressed (Fig. 2D). In about half the ovarian tumors examined, PGRMC1 was predominantly detected in the cytoplasm, whereas in the remaining tumors, PGRMC1 was observed throughout the cell including the nucleus (Fig. 2E). To confirm this observation, the cellular localization of PGRMC1 was assessed using confocal microscopy. This microscopic approach confirmed that PGRMC1 was predominantly cytoplasmic in some tumors (Fig. 3A) and localized throughout the cell in others (Fig. 3B). In many of these tumor cells, PGRMC1 appeared to be highly concentrated in the nucleus (Fig. 3B).
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). The mRNA that encodes PGRMC1 was detectable by RT-PCR in Ovcar-3 cells (Fig. 4A). PGRMC1 expression was also confirmed by Western blot as revealed by the presence of a protein band of approximately 66 kDa, which likely represents a trimer of the 22-kDa PGRMC1 molecule (Fig. 4B). This assessment is based on the finding that exposure to high concentrations of dithiothreitol is required to disrupt the disulfide bridges that bind monomers of PGRMC1 together (29). PGRMC1 was observed throughout the cytoplasm and nucleus of Ovcar-3 cells (Fig. 4C). PGRMC1 also appeared to be preferentially localized to either the cytoplasm or nucleus, depending on the individual cell (Fig. 4C). Finally, PGRMC1 was detected along a limited area of plasma membrane (Fig. 4C).
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). In the presence of 10% fetal bovine serum, CDDP induced a dose-dependent increase in the percentage of Ovcar-3 cells that undergo apoptosis (Fig. 5B). This effect of CDDP was attenuated at both 5 h (P < 0.05) and 24 h (P < 0.05) of culture by simultaneous treatment with P4 (Fig. 5C).
To determine the role of PGRMC1 in mediating P4’s antiapoptotic action, Ovcar-3 cells were transfected with a GFP-PGRMC1 expression vector. As can be seen in Fig. 6A, transfection with the GFP-PGRMC1 vector increased the level of PGRMC1. In addition, GFP-PGRMC1 was expressed in about 30% of the cells (Fig. 6B). In the presence of P4, Ovcar-3 cells transfected with GFP-PGRMC1 were less responsive to the apoptotic effects of CDDP, compared with those Ovcar-3 cells transfected with empty GFP vector alone (P < 0.05; Fig. 6C).
PGRMC1 levels were also depleted by treatment for 72 h with three different PGRMC1 siRNAs. As seen in Fig. 7A, each of the PGRMC1 siRNA suppressed PGRMC1 mRNA levels, compared with the scramble control. Similarly, proteins levels of PGRMC1 were reduced by PGRMC1 siRNA treatment (Fig. 7B). Immunocytochemical analysis after 72 h treatment with either scramble control or PGRMC1 siRNA (ID no. 18248) confirmed the effectiveness of this siRNA treatment to suppress PGRMC1 levels (compare Fig. 8A with Fig. 8B). As might be expected, this PGRMC1 siRNA treatment attenuated P4’s ability to inhibit CDDP-induced apoptosis (P < 0.05; Fig. 8C). Similarly, treatment with PGRMC1 blocking antibody reduced the capacity of P4 to inhibit the apoptotic effects of CDDP (Fig. 8D).
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It is interesting that there exists an inverse relationship between PGR and PGRMC1 expression. This is consistent with the idea that PGR activation suppresses PGRMC1 expression. This concept is supported by the finding that P4 suppresses PGRMC1 levels and that PGRMC1 levels are increased in PGR null mice (30). Although an analysis of the PGRMC1 promoter failed to reveal the presence of a P4 response element, a consensus glucocorticoid response element (GRE) was detected (31). Because P4 can mediate its transcriptional activity through a GRE (32), it is conceivable that ligand-activated PGR acting through the GRE could antagonize the expression of PGRMC1. Therefore, the protective effect of P4 may be due in part to PGR mediated suppression of PGRMC1 because lowering the levels of PGRMC1 would increase the sensitivity of ovarian cancer cells to CDDP (see subsequent discussion).
In all ovarian cancer cells, PGRMC1 localizes to the cytoplasm and plasma membrane, but in some of these cells, PGRMC1 appears to concentrate in the nucleus. PGRMC1 is also observed within the nucleus of some Ovcar-3 cells. In HeLa cells PGRMC1 has been shown to localize to the nucleus if it is phosphorylated at two sites (serine 57 and 181) (33). Both of these serines are within consensus casein kinase 2 sites (see http://www.expasy.org/uniprot/O00264). This implies that PGRMC1 localization is highly and precisely regulated possibly through a kinase cascade that involves casein kinase 2. The physiological significance of nuclear PGRMC1 is unknown but might reflect a transcriptional function because our microarray studies indicated that PGRMC1 siRNA treatment alters the mRNA levels of 80 genes more than 1.5-fold (our unpublished observations). Whether the expression of any of these genes is directly regulated by nuclear PGRMC1 is unknown, but this finding is consistent with a proposed transcriptional function for nuclear PGRMC1.
Clearly the ratio of PGRMC1 to PGR is changed in the different stages of cancer, and the ratio of these two P4 receptors could change the tumor’s response to P4. Interestingly, in vitro studies have shown that P4 affects the growth of ovarian cancer cell lines in a biphasic manner (4, 34, 35, 36). At low concentrations (i.e. nanomoles), P4 promotes growth, whereas at higher concentrations (micromoles), P4 inhibits growth and prevents apoptosis (4, 34, 35, 36). In cell lines that do not express PGR, the growth-promoting actions are not observed (37), suggesting that the proliferative effect of P4 is mediated by PGR. This ability to respond to low levels of P4 is consistent with the PGR’s P4 binding characteristics (i.e. dissociation constant = 1–5 nM) (38) and the findings that ligand activation of PGR stimulates MAPK activity and genes that are required for cell proliferation (39). Ironically, the P4-induced increase in proliferation may make the cells more susceptible to CDDP because CDDP binds to the DNA in mitotic cells (40, 41).
Given the expression pattern of PGR and PGRMC1, it is important to determine P4’s effects on ovarian cancer cells where the ratio of PGRMC1 to PGR dramatically increases as it does in more advanced-stage ovarian cancers. The present studies demonstrate that P4 treatment of Ovcar-3 cells that express PGRMC1 but not PGR promotes cell survival and resistance to CDDP. Therefore, determining the ratio of PGRMC1 to PGR mRNA may be useful as a biomarker to predict the ovarian tumor’s response to P4 and sensitivity to CDDP.
Although the descriptive observations presented in this paper suggest a role of PGRMC1 in regulating ovarian cancer viability, they do not conclusively demonstrate that PGRMC1 is required for P4’s antiapoptotic action. To establish this causal relationship, the level of PGRMC1 was modulated and P4 responses monitored in a well-characterized human ovarian cancer cell line, Ovcar-3 cells (i.e. Ovcar-3 cells have more than 500 citations in the PubMed database). These cells have been shown to be responsive to P4 (34), and previous studies indicated that they express PGR (42). However, like ovarian cancers, the receptor status of ovarian cancer cell lines can change. The RT-PCR analysis in the present study failed to detect PGR in the Ovcar-3 cells, which were provided by Dr. Nellie Auersperg (University of British Columbia). RT-PCR analysis did detect PGRMC1. The presence of PGRMC1 was also confirmed by Western blot analysis. Because these Ovcar-3 cells express PGRMC1 but not PGR, they mimic the P4 receptor status of more advanced ovarian cancers and thereby serve as a good model to study PGRMC1’s role in the more advanced ovarian cancers.
In normal human and rodent ovarian cells, P4 inhibits apoptosis (19, 20, 25, 28). The present studies also show that P4 inhibits Ovcar-3 cells from undergoing apoptosis induced in response to serum withdrawal with a maximum effect in the 1-µM range. Whereas this amount of P4 is higher than that normally observed in serum, ovarian levels of P4 are severalfold greater than 1 µM (35). Moreover, ovarian cancer cells can synthesize P4 (7), thus ensuring that the ovarian cancer cells are continuously in a high P4 environment. This high P4 environment likely promotes ovarian cancer cell viability and decreases their sensitivity to CDDP. This is supported by the present in vitro studies that demonstrate that CDDP in the presence of serum induces apoptosis in 40–60% of the Ovcar-3 cells within 24 h. However, the addition of P4 reduces the killing effect of CDDP by about 40%.
That the antiapoptotic action of P4 in Ovcar-3 cells is mediated by PGRMC1 is supported by three different experimental approaches. First, overexpression of PGRMC1, which is known to increase the number of cellular binding sites for 3H-P4 (28), reduces the killing effects of CDDP by about 50%. Second, depleting PGRMC1 with siRNA treatment enhances the killing effect of CDDP in the presence of P4 by 50% or more. Third, an antibody to PGRMC1 makes the cells more sensitive to CDDP, even in the presence of P4. The observed effect of the antibody treatment is consistent with previous work with normal ovarian cells (19, 25, 28) and implies that P4 triggers an antiapoptotic signal transduction pathway, in part through a membrane-initiated event, because antibodies do not penetrate the cell membrane. However, PGRMC1 also localizes to the cytoplasm and nucleus. It is possible then that PGRMC1 may act not only at the plasma membrane but also within the cytoplasm and nucleus to regulate several different pathways with each pathway being essential for cell survival. Regardless of the precise site of action, these in vitro studies provide compelling evidence that PGRMC1 promotes ovarian cancer cell viability. More importantly, they also provide a scientific basis for the development of an adjunct therapy for the treatment of ovarian cancers because both antibodies (43) and more recently siRNAs (44, 45, 46) have been used to target specific cancers and/or oncogenic proteins. This concept is presently being tested by establishing human ovarian tumors in nude mice.
In summary, the present study demonstrates that ovarian cancers express PGRMC1 and that its activation promotes the survival of these cells and makes them more resistant to CDDP. These observations suggest the PGRMC1 may be a good protein to target for development as a biomarker. Moreover, the finding that either siRNA or PGRMC1 antibody treatment attenuates PGRMC1’s actions indicates that these experimental approaches have the potential to be used clinically to ablate PGRMC1 and thereby improve the efficiency of CDDP therapy.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online March 4, 2008
Abbreviations: CDDP, Cisplatin; DAPI, 4',6'-diamino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GRE, glucocorticoid response element; P4, progesterone; PGR, progesterone receptor; PGRMC1, PGR membrane component-1; siRNA, small interfering RNA.
Received December 19, 2007.
Accepted February 27, 2008.
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