Skip Navigation


Annals of Oncology Advance Access originally published online on January 10, 2008
Annals of Oncology 2008 19(4):696-705; doi:10.1093/annonc/mdm557
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/4/696    most recent
mdm557v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Thasni, K. A.
Right arrow Articles by Priya, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thasni, K. A.
Right arrow Articles by Priya, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

gynecologic tumors

Estrogen-dependent cell signaling and apoptosis in BRCA1-blocked BG1 ovarian cancer cells in response to plumbagin and other chemotherapeutic agents

K. A. Thasni1, S. Rakesh1, G. Rojini1, T. Ratheeshkumar1, G. Srinivas2 and S. Priya1,*

1 Laboratory of Molecular Therapeutics, Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Thiruvananthapuram, Kerala
2 Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, Kerala, India

* Correspondence to: Dr S. Priya, Division of Cancer Biology, Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Thiruvananthapuram 695 014, Kerala, India. Tel: +91-471-2347975; Fax: +91-471-2348096; E-mail: priyasrinivas2000{at}yahoo.com


   Abstract
Top
 Abstract
introduction
 experimental procedures
 results
 discussion
 funding
 Acknowledgements
 References
 
Background: Cellular response to chemotherapeutic drugs in the absence of BRCA1 either completely or partially had drawn less attention. The present study evaluated whether there is a differential inhibition of cell growth by selected compounds with respect to BRCA1 status in estrogen receptor (ER)-positive ovarian cancer cells.

Materials and methods: The BG1 ovarian cancer cells used in the experiments were antisensely blocked with BRCA1 gene. Growth inhibition and apoptotic induction were analyzed to evaluate the cytotoxic effects. Small interfering RNA (SiRNA) transfection, western blot analysis, RT–PCR analysis and molecular modeling were carried out to analyze the estrogen-dependent action of plumbagin.

Results: Although we found that all the compounds studied induce apoptosis, the induction was in the order of plumbagin > doxorubicin > tamoxifen > cisplatin. Plumbagin can bind to the active site of ER-{alpha}. Plumbagin, however, induced ER-{alpha} 46 kDa truncated isoform, which was found abundantly preempted in the cytoplasm compared with a 66-kDa full-length isoform. The truncated isoform is known to inhibit classical ER-{alpha} signaling pathways. SiRNA-transfected cells for ER-{alpha} exhibited lower cytotoxicity upon plumbagin treatment than the control-transfected cells.

Conclusion: Taken together, this study indicates that plumbagin has chemotherapeutic potential in BRCA1-mutated/defective ER-positive cancers.

Key words: BRCA1, cisplatin, doxorubicin, plumbagin, tamoxifen


   introduction
Top
 Abstract
 introduction
experimental procedures
 results
 discussion
 funding
 Acknowledgements
 References
 
We had earlier found increased propensity for apoptosis in BRCA1-blocked cells (BG1, ovarian cancer cells) to plumbagin, a plant-derived napthaquinone [1]. Since BRCA1 is involved in estrogen receptor (ER) inhibition, an increased susceptibility for these cells to antiestrogen drug, tamoxifen (TAM) was anticipated. We found that plumbagin, however, was more effective in inducing apoptosis than TAM [1]. Further, it was demonstrated that the mode of action of plumbagin was through a reactive oxygen species (ROS)-dependent mechanism [2]. At present, there is no difference in the treatment strategy for BRCA1-mutated patients, even though accumulating evidences indicate that it can be done [3].

The scheduled study is pursued by using chemotherapeutic agents such as cisplatin (CIS) and doxorubicin (DOX) in comparison to plumbagin, which are known to induce (DNA damage-mediated cell growth) inhibition of tumor cells. We have analyzed whether these compounds activate p53 (whether apoptosis induction involves p53), BARD1 (BRCA1 ring domain 1, which is a binding partner of BRCA1) and ER-{alpha} (because these cells are ER-{alpha} overexpressing).

The results of this study indicate that plumbagin is more effective (at a lower concentration) than CIS/DOX/TAM in inducing apoptosis in BRCA1-blocked cells (ER-positive ovarian cancer cells) than BRCA1-unblocked cells. Plumbagin may interfere with ER-{alpha} signaling pathway and induce apoptosis in a death receptor-mediated pathway.


   experimental procedures
Top
 Abstract
 introduction
 experimental procedures
results
 discussion
 funding
 Acknowledgements
 References
 
cell lines
BG1 cells derived from a patient with stage III, poorly differentiated ovarian adenocarcinoma is an estrogen-responsive cell line with wild-type BRCA1 gene. Antisense-blocked cells (from exons 1 to 11) were termed as AS4 and control vector-transfected cells were termed NEO [2, 4]. BRCA1 repression with antisense blocking causes partial reduction in the protein expression which can be comparable to BRCA1-mutated condition where functional protein is not produced. The cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), with 1x antibiotic antimycotic (Invitrogen, NY). Before each experiment, the cells were pretreated for 5 days in phenol red-free, DMEM/F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% charcoal/dextran-treated serum (Hyclone, Logan, UT) and 10–8 M 17β-estradiol [1,3,5(10)-estratriene 3,17β-diol; Sigma-Aldrich, St Louis, MO].

chemicals
DOX, CIS and TAM citrate were obtained from Sigma-Aldrich. Plumbagin used in the experiments was isolated from the Indian medicinal plant, Plumbago zeylanica as mentioned elsewhere [5]. Annexin staining kit and mouse monoclonal antibodies to ER-{alpha} (D-12, sc-8005) were purchased from Santa Cruz Biotechnology Inc. (Santacruz, CA) and ER-β (E1276) and p53 (P5813) from Sigma-Aldrich. The ApoAlertTM Mitochondrial Membrane Sensor Kit was purchased from Clontech (Palo Alto, CA). The spectroflourimetric assay substrates for caspase-3, -6 and -8, AcDEVD-AFC, AcVEID-AFC and AcIEHD-AFC, respectively, were bought from BD PharMingen, BD Biosciences, San Jose, CA. 3-(4,5-Dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from USB Amersham Biosciences (Cleveland, OH).

[3H]thymidine incorporation
Thymidine incorporation was carried out to determine the growth differences of the AS4 and NEO cells. For [3H]thymidine incorporation, cells were treated with [3H]thymidine for 6 h (0.5 µCi per well), and proteins were precipitated in ice-cold 5% trichloroacetic acid (100 µl per well). The cells were solubilized with 0.2 N NaOH. The radioactivity was counted using a liquid scintillation counter. The extent of thymidine incorporation was the measure of the growth proficiency of the cells.

MTT assay
This assay was done as described elsewhere [2]. The drugs DOX and CIS were added at different concentrations in quadruplicate samples. A concentration lower than the LD50 was used for further experiments. The concentrations used for further experimentations were plumbagin (2.5 µM), TAM (18.5 µM), DOX (15 µM) and CIS (200 µM).

Annexin V binding
Annexin V-fluorescein isothiocyanate (FITC) (200 µg/ml) was used for staining as described previously [2]. The apoptotic cells were viewed using LEICA DIMRE-2 inverted confocal laser scanning microscope equipped with Ar/He/Ne laser (488 nm) with x20 magnification green filter (tetra methyl rhodamine isothiocyanate 540 nm) and photographed.

assessment of mitochondrial membrane potential {Delta}{psi}m
This assay was done with ApoAlertTM Mitochondrial Membrane Sensor Kit (Clontech) [1]. The cells were viewed under confocal laser scanning microscope (488 and 540 nm) and photographed.

caspase activation
The caspase-3, -6 and -8 activity was assessed spectrofluorimetrically (Perkin Elmer LS-50, Waltham, MA) (excitation and emission wavelengths 400 and 505 nm, respectively) as described [6].

analysis of p53, BARD1 and RAD51 expression profile by RT–PCR
Total RNA was isolated from the cells using TRIZOL reagent (Invitrogen). For cDNA isolation, 2 µg/µl of total RNA and random hexamers were incubated at 70°C for 2 min. To the reaction, 1x Moloney Murine Leukemia Virus (MMLV) RT buffer, 1 mM deoxy nucleotide triphosphate 200 U of MMLV RT and 10 U of RNasin Ribonuclease inhibitor were added to make up the final reaction volume to 30 µl and incubated at 42°C for 1.5 h, 4°C for 2 min and 70°C for 2 min. All reagents were from Promega Corporation (Madison, WI) except when otherwise specified. Gene-specific primers are listed in Table 1.


View this table:
[in this window]
[in a new window]

  		Table 1. The primer pairs used in this study for RT–PCR

 
The PCR profile for p53 was 95°C for 1 min, 60°C for 2 min and 72°C for 2 min; and BARD1 95°C for 10 s, 60°C for 30 s and 72°C for 1.3 min and for β-actin the cycle condition was 95°C for 30 s, 50°C for 30 s and 72°C for 45 s. The products of amplification were resolved in a 2% agarose gel and photographed using a gel documentation system.

western blotting for ER
The whole-cell lysate was isolated as described before [1]. Cytosolic and nuclear extracts were isolated and 50 µg of protein was loaded for western blots and probed with respective antibodies.

nuclear lysate preparation
After incubating the cells with the compounds for 1 h at 37°C, the cytosolic and nuclear extracts were isolated using the cytosolic extraction buffer [solution A (1 M Hepes, 2 M KCl, 0.1 M EGTA) (0.976 µl), 0.1 M dithiothreitol (DTT) (10 µl), 100 mM PMSF (5 µl), 1 mg/ml leupeptin (2 µl), 1 mg/ml aprotinin (2 µl) and 250 mg/ml benzamidine (2 µl)] and the nuclear isolation buffer [solution B (1 M Hepes, 5 M KCl, 0.1 M ethylene glycol tetra acetic acid (EGTA)) (0.976 µl) (0.1 M DTT(10 µl), 100 mM phenyl methyl sulfonyl fluoride (PMSF) (10 µl), 1 mg/ml leupeptin (2 µl), 1 mg/ml aprotinin (2 µl) and 250 mg/ml benzamidine (2 µl)]. For western blot, 50 µg protein were loaded.

transient transfection studies with siRNA
A 21–23 bp siRNA [ER-{alpha} shortcut siRNA mix, New England Biolabs, Inc. (NEB) Ipswich, MA] was used for silencing ER-{alpha}. Cells were cultured in 96-well plates in 200 µl DMEM/Ham’s F-12 medium supplemented with 10% FBS. After 24 h, siRNA was transfected (20 nM per well) using TransPass R1 transfection reagent (NEB). Posttransfected cells (48–50 h) were treated with plumbagin (2.5 µM), TAM (18.5 µM) and CIS (200 µM) and after 24 h cell viability was carried out by MTT assay as described earlier.


   results
Top
 Abstract
 introduction
 experimental procedures
 results
discussion
 funding
 Acknowledgements
 References
 
plumbagin is most effective in cell growth inhibition than CIS, DOX or TAM in BRCA1-blocked cells
We confirmed BRCA1 gene silencing by western blot analysis (data not shown) and determined the growth advantage of BRCA1 antisense (AS4) cells over control (NEO) cells by [3H]thymidine incorporation assay. AS4 cells incorporated up to five-fold more [3H]thymidine than NEO cells, indicative of their higher proliferative nature (Figure 1A).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. (A) Cells were treated with [3H]thymidine (0.5 µCi per well) and incubated for 6 h. The extent of thymidine incorporation indicated the growth proficiency of the cells. (B and C) Cell viability was assessed in AS4 and NEO cells using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay with both cisplatin and doxorubicin.

 
Differential inhibition of cell growth by the above agents was analyzed by growth inhibitory MTT assay. Both AS4 and NEO cells were less sensitive to CIS for a wide range of concentrations as evident from Figure 1B. The calculated LD50 value for DOX was 15 and 25 µM for AS4 and NEO cells, respectively (Figure 1C). The calculated LD50 value for CIS was >200 µM for both AS4 and NEO cells (Table 2), but that of plumbagin and TAM were as reported earlier [1]. These results indicate that plumbagin was most effective in inducing cell growth inhibition when compared with CIS, DOX or TAM.


View this table:
[in this window]
[in a new window]

  		Table 2. LD50 values for the drugs tested

 
plumbagin induces apoptosis more effectively than TAM, CIS and DOX in BRCA1-blocked cells
Flip-flop of phosphatidyl serine to the outer surface of the membrane represents early stage of apoptosis and was detected as bright green fluorescence when stained with annexin V-FITC. No significant difference, in annexin binding, was observed between AS4 and NEO cells, on treatment with CIS, DOX and TAM. In contrast, the cells treated with plumbagin showed more late apoptotic changes in AS4 cells than NEO cells (Figure 2A). Propidium iodide (PI) staining showed late apoptotic behavior in treated cells.


Figure 2
View larger version (96K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. (A) Annexin propidum iodide staining using confocal microscopy (CON, control; PB, plumbagin; TAM, tamoxifen; DOX, doxorubicin; CIS, cisplatin). (B) Analysis of loss of mitochondrial membrane potential ({Delta}{psi}m).

 
One of the initial events in apoptosis is loss of mitochondrial membrane potential. MitoSensorTM gets aggregated in the mitochondria of healthy cells and fluoresce red. In contrast, when mitochondrial membrane potential is lost it shows only green fluorescence due to the presence of unpolymerized dye in the cytoplasm. The number of cells, which showed green fluorescence, was less in the case of CIS and DOX when compared with cells treated with plumbagin and TAM. The extent of loss of mitochondrial membrane potential was more in plumbagin-treated AS4 cells than NEO cells (Figure 2B).

Caspases are a family of cysteine proteases that are activated during different phases of the apoptosis and once activated, they cleave and activate downstream caspases. As shown in the Figure 3A, drug treatment in AS4 cells activated all the three caspases (6, 3 and 8) at 6 h. There was no statistically significant difference between the caspase activities of AS4/NEO when treated with CIS/DOX/TAM at 6 h. DOX showed a relatively increased caspase-3 activity than caspase-6 and -8 activities at 6 h. Caspase activity (at 6 h) was found to be higher in AS4 cells than in NEO when treated with plumbagin, even though the mean difference never reached statistical significance. At 12 h, only caspase-8 showed ~6.5-fold increase for plumbagin-treated AS4 cells (Figure 3B).


Figure 3
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Plumbagin induces apoptosis through caspase-8 activation. (A) Caspase activation (6, 3 and 8) by the compounds for 6 h. (B) Caspase activation (6, 3 and 8) by the compounds for 12 h. Caspase activity was expressed as fold activation over control.

 
plumbagin-induced p53 and BARD1 expression is dependent on the presence of estradiol
All the four compounds increased p53 mRNA (messenger RNA) levels in NEO cells at 1.5 h as shown in Figure 4A and B. In contrast, in AS4 cells these drugs reduced p53 mRNA levels. In the absence of estradiol, p53 mRNA expression however, was high in AS4 control cells as well as when treated with plumbagin or TAM in comparison to NEO cells (Figure 5A). RT–PCR results point to the fact that BARD1 expression was also high in AS4 cells when treated with plumbagin at 4 h and was estrogen dependent (Figure 5B). Basal level expression of p53 and ER-{alpha} was high in AS4 cells (Figure 6). These results indicate that plumbagin-induced cell death was enhanced in the absence of estradiol, which may be because the cells are estrogen responsive.


Figure 4
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. (A) RT–PCR analysis for BRCA1 ring domain 1 (BARD1), p53 and β-actin in plumbagin, tamoxifen, doxorubicin, cisplatin and control-treated AS4 and NEO cells (1.5 h treatment). (B) Expression values for each PCR product were normalized to their respective β-actin expression by Quantity One software (Bio-Rad, Hercules, CA).

 

Figure 5
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. (A) RT–PCR analysis for BRCA1 ring domain 1 (BARD1), p53 and β-actin in the presence and absence of 17β-estradiol (4 h treatment) (B) Expression values for each PCR product were normalized to their respective β-actin.

 

Figure 6
View larger version (60K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. Western blot analysis showing basal level expression for estrogen receptor (ER)-{alpha} and p53 in AS4 and NEO cells.

 
plumbagin causes increased expression of 46 kDa truncated ER-{alpha} isoform in the cytoplasm than TAM
In order to further explore the expression of ER, we investigated the expression of ER-{alpha} in nuclear and cytosolic extracts after plumbagin treatment. It was found that a truncated form of ER-{alpha} was expressed more in the cytoplasm of plumbagin-treated AS4 cells, in comparison to cells treated with TAM (Figure 7A). Cytosolic and nuclear extracts of plumbagin-treated cells show abundant ER-{alpha} 46 kDa protein expression, however, ER-β expression does not seem to be changed in response to plumbagin (Figure 7B and C). Higher expression of ER-{alpha} 46 kDa isoform reveals that plumbagin induces apoptosis by activation of this isoform, which is known to inhibit classical ER-{alpha} signaling pathway [7]. RAD51, which is a binding partner of BRCA1, was found to be decreased in BRCA1-blocked cells treated with plumbagin (Figure 7D).


Figure 7
View larger version (69K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 7. (A) Western blot analysis for estrogen receptor (ER)-{alpha} in AS4 and NEO cells after treatment with plumbagin (PB), cisplatin (CIS) and tamoxifen (TAM) for 1.5 and 4 h. The result was normalized using β-actin. (B) Western blot analysis for ER-{alpha} with nuclear and cytoplasmic isolate in PB-treated AS4 and NEO cells. (C) Western blot analysis of ER-β protein in AS4 and NEO cells after treatment with PB and TAM. (D) RT–PCR demonstrating the expression of RAD51, hER-{alpha} 66 kDa full-length transcript and truncated form (ER-{alpha}, FF and ER-{alpha}, AF) after PB treatment. Forward primers for RT-PCR were based on sequence in 5' exons 1A & 1F , and the reverse primer based on sequence in exon 2 (common to both reactions). {Delta}1a-hER{alpha}-46 transcripts are derived from the 5' exon 1F which is referred to as FF (first F referred to as exon 1F and second F denotes forward). Full length hER{alpha}-66 transcripts are derived from the 5' exon 1A which is referred to as AF (A referred to as exon 1A and second F denotes forward).

 
The expression of ER-{alpha} transcripts in response to plumbagin was studied by RT–PCR. Both truncated and full-length ER-{alpha} transcripts were present in control cells when amplified by primers corresponding to exon 1F region at C-terminal end of ER-{alpha}. When amplified by primers specific to exon 1A region, which will be spliced from 5' untranslated region resulting in human estrogen receptor (hER-{alpha}) 46 kDa transcript, we observed corresponding bands in plumbagin-treated cells (Figure 7D).

Molecular docking studies using docking tools (Gold 2.12 Academic Version) to identify whether there is any prospective interaction between plumbagin and ER-{alpha} which revealed that plumbagin may bind to ER-{alpha} with higher affinity to the active site where estradiol bind (with a Gold Score of 35) (Figure 8).


Figure 8
View larger version (46K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 8. Proposed model for plumbagin's binding in the active site of estrogen receptor-{alpha}. Molecular docking tools (Gold 2.12 Academic Version) revealed that plumbagin bind to ER-{alpha} with higher affinity to the active site where estradiol can bind (with a Gold Score of 35).

 
siRNA-mediated repression of ER-{alpha} decrease the effect of plumbagin
To establish ER-{alpha} dependence for plumbagin action in AS4 and NEO cells, growth inhibitory assay was carried out in ER-{alpha} siRNA-transfected cells. In response to plumbagin, cell death was decreased in ER-{alpha} siRNA-transfected cells compared with untransfected cells (Figure 9), indicating that the depletion of ER-{alpha} expression decreases plumbagin-mediated cell growth inhibition. In the NEO cells, ER-{alpha} blocking completely protected the cells from cytotoxic effects of plumbagin treatment. TAM was more effective in ER-{alpha}-positive AS4 and NEO cells as expected. When both ER-{alpha} and BRCA1 were blocked, the effect of TAM is further reduced indicating that TAM will not be effective in ER-negative BRCA1-mutated cells. Cells remained insensitive to CIS, irrespective of ER-{alpha} and BRCA1 status. The above results indicate that plumbagin has specific growth inhibitory activity in BRCA1-blocked ER-{alpha} overexpressed condition.


Figure 9
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 9. Effect on cell growth by estrogen receptor (ER)-{alpha} siRNA-mediated inhibition on (A) AS4 and (B) NEO cells. Posttransfected cells (48–50 h) were treated with plumbagin (2.5 µM), tamoxifen (18.5 µM) and cisplatin (200 µM). After 24 h, cell viability was carried out using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay as described earlier.

 

   discussion
Top
 Abstract
 introduction
 experimental procedures
 results
 discussion
funding
 Acknowledgements
 References
 
BRCA1, a protein involved in DNA repair and apoptosis, its mutation results in defective cellular functions. Genomic instability resulting in mutations in the other genes cause higher rates of tumorigenesis in BRCA1 mutation carriers [8]. Several lines of evidence indicate a cardinal role for steroid hormones and their receptors in the genesis of BRCA1 mutant cancers. BRCA1 overexpression can inhibit estrogen and progesterone-stimulated cell proliferation in vitro [9, 10]. BRCA1 has been proved to physically interact with ER-{alpha} and inhibit its transcriptional activity. Thus, one of the major mechanisms of tumorigenesis in BRCA1-mutated cancers may be through increased activity of ER-{alpha} rather than its overexpression. This may be the reason why most BRCA1-related cancers are immunohistochemically negative for ER-{alpha} and progesterone receptor. We had earlier reported that plumbagin is more effective in inducing apoptosis than TAM in AS4 cells [1]. Since sensitivity of BRCA1-deficient cells to DOX is highly controversial [11, 12] and CIS is reported to regulate the expression of BRCA1 [13], a comparative study was done to decipher the effect of CIS/DOX with respect to plumbagin/TAM in BRCA1-blocked BG1 ovarian cancer cells.

There was no specific cell growth inhibitory capacity for BRCA1 blocked cells in comparison to NEO cells in response to CIS, a cross-linker that can interfere with DNA repair pathways.

BARD1 expression was not altered by CIS treatment irrespective of BRCA1 status, indicating that BARD1-induced apoptosis is not related to CIS response. Hence, it can be indicated that BG1 ovarian cancer cells are resistant to CIS, irrespective of BRCA1 status.

DOX, an anthracycline class of anticancer drug generates free radicals that induce DNA strand breaks thereby interfering in the DNA repair pathways. Our results show that in comparison to CIS, DOX was more sensitive to BRCA1-blocked cells with ~1.7-fold increase in growth inhibition than NEO cells. Annexin staining and loss of mitochondrial membrane potential reveal the efficacy of DOX in inducing apoptosis in BRCA1-blocked cells. The level of caspase-3 was found to be increased two-fold at 6 h when compared with caspase-6 and -8 in response to DOX treatment.

TAM, a well-known antiestrogen, did not show a difference in its sensitivity towards BRCA1-blocked cells [1]. Apoptosis-inducing capacity was higher for TAM, when collated to CIS. Zhang et al. [14] reported that TAM-induced apoptosis in breast cancer cells are without alteration of p53 protein levels. At 4 h in the absence of estrogen, p53 expression was high in control as well as in plumbagin- and TAM-treated BRCA1-blocked cells.

There was no significant increase in BARD1 expression in response to TAM in AS4 cells. The result points to the fact that there was no much difference in induction of apoptosis in BRCA1-blocked or -unblocked cells, however, the mode of action of TAM-induced apoptosis is different with respect to BRCA1 status.

The LD50 values were less for the BRCA1-blocked cells when compared with NEO cells with plumbagin as already reported (Table 2). Plumbagin treatment triggers the mitochondrial apoptotic pathway indicated by mitochondrial membrane potential loss and caspase-9 activation [1, 15]. In BRCA1-blocked plumbagin-treated cells, caspase-8 activity was increased to ~6.5-fold, indicating that BRCA1 blockage sensitizes BG1 cells to plumbagin more effectively and induces apoptosis mainly by death receptor pathway. We found that irrespective of BRCA1 status all the drugs induced apoptosis in the order plumbagin > DOX > TAM > CIS as evidenced by MTT, PI staining, annexin binding and loss of mitochondrial membrane potential. BARD1 expression was increased to ~nine-fold in BRCA1-blocked cells, which was highly dependent in the presence of estradiol in response to plumbagin. BARD1 induces apoptosis by catalyzing phosphorylation of p53 by DNA damage response kinase [16]. It has also been reported that BARD1 has preapoptotic functions independent of its association with BRCA1 [17]. Therefore, BARD1 induction occurred as a result of plumbagin treatment and may be one of the mechanisms by which it induces apoptosis in BRCA1-deficient cells. RAD51 is reported to be cleaved by caspase-3 during apoptosis [18]. The decrease in the RAD51 expression in BRCA1-blocked cells indicates that caspases 3 cleavage is more active in these cells in response to plumbagin. Blockade of p53 partially decreased plumbagin-induced apoptosis [15]. In our study, we found that when BRCA1 is blocked, p53 expression is increased which may result in increased sensitivity to plumbagin compared with NEO cells.

We have also detected a 46-kDa protein that is present abundantly than the full-length 66 kDa ER-{alpha} protein by western blotting in plumbagin-treated cells. This shorter isoform is reported to be generated by alternative splicing of the 66-kDa ER-{alpha}. RT–PCR analysis also revealed that the full-length ER-{alpha} is absent when treated by plumbagin in both AS4 and NEO cells. Human ER-{alpha} 46 kDa is a powerful inhibitor of hER-{alpha} 66 kDa in a cell context where the transactivation function of AF-1 predominates over AF-2. This truncated ER-{alpha} acts as a competitive inhibitor of hER-{alpha} 66 kDa-mediated transactivation [19]. Western blot analysis of ER-β expression does not seem to be varied much by plumbagin or TAM treatment (Figure 7C).

It was also shown that siRNA-mediated repression of ER-{alpha} decrease the effect of plumbagin. In the BRCA1-unblocked cells, ER-{alpha} blocking almost completely recovers the decreased viability by plumbagin treatment which indicates that plumbagin has specific growth inhibitory activity in BRCA1-blocked ER-{alpha} overexpressed cells.

To conclude, even though CIS, DOX and TAM can cause a growth inhibitory effect on BRCA1-blocked ER-positive ovarian cancer cells; they cannot be used specifically for ER-{alpha} positive BRCA1-mutated cancers, whereas plumbagin will be a more effective chemotherapeutic agent that can be used for the above-mentioned type of cancers. This may be due to at least three reasons as depicted in Figure 10.


Figure 10
View larger version (47K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 10. A proposed model for the action of plumbagin in BRCA1-blocked ovarian cancer cells. Single-headed arrow indicates activation and closed heads indicate inhibitory pathways.

 
(i) The generation of ROS by plumbagin can create DNA damage [2]. BRCA1 may be physiologically important for mounting normal response to xenobiotic insults and oxidative stress [20–22]. (ii) Inhibiting ER signaling pathway by induction of truncated ER-{alpha} 46 kDa isoform which acts as a competitive inhibitor of hER-{alpha} 66 kDa mediated transactivation. BRCA1 is known to inhibit ER-{alpha} activation [23]. (iii) Plumbagin down-regulated the expression of angiogenic factor [vascular endothelial growth factor (VEGF)] [24]. N-terminus of BRCA1 is essential for binding to ER-{alpha} and for down-regulating VEGF transcription via the estrogen signaling pathway [25]. In addition to this, our own results show that RAB18, M41 and certain heat shock proteins were activated by plumbagin in BRCA1-blocked cells (K. A. Thasni, T. Ratheesh Kumar, G. Rojini et al., unpublished data). The above cellular effects due to plumbagin would have normally been taken care of by the native BRCA1 protein, which is absent in AS4 cells resulting in increased apoptosis. This throws light on the fact that plumbagin has chemotherapeutic potential as an anticancer agent in estrogen-responsive BRCA1-mutated ovarian cancer patients. More research, however, has to be done to elucidate cell signaling pathways induced by plumbagin with respect to BRCA1 status.


   funding
Top
 Abstract
 introduction
 experimental procedures
 results
 discussion
 funding
Acknowledgements
 References
 
Department of Science and Technology (SR/FT/L-111/2003), Department of Biotechnology (BT/R&D/15/27/95-RGCB); Government of India, Kerala state Council for Science and Technology Environment [(T) 028/SRS/2005/CSTE] to P.S.


   Acknowledgements
Top
 Abstract
 introduction
 experimental procedures
 results
 discussion
 funding
 Acknowledgements
References
 
We thank Lois Annab, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA, for the generous gift of AS4 and NEO cells. We also acknowledge Asoke Banerji, Centre for Biotechnology, Amrithapuri Campus, Clappana PO, Kollam 690525, Kerala, India, for isolating and purifying plumbagin for this study. We also acknowledge H. Ramkumar for helping in molecular docking studies. The authors (TKA and SR) also acknowledge the research fellowship by Council for Scientific and Industrial Research, Government of India.

Received for publication April 17, 2007. Revision received September 19, 2007. Revision received November 6, 2007. Accepted for publication November 7, 2007.


   References
Top
 Abstract
 introduction
 experimental procedures
 results
 discussion
 funding
 Acknowledgements
 References
 
1. Srinivas G, Annab LA, Gopinath G, et al. Antisense blocking of BRCA1 enhances sensitivity to plumbagin but not tamoxifen in BG-1 ovarian cancer cells. Mol Carcinog (2004) 39:15–25.[CrossRef][Web of Science][Medline]

2. Srinivas P, Gopinath G, Banerji A, et al. Plumbagin induces reactive oxygen species, which mediate apoptosis in human cervical cancer cells. Mol Carcinog (2004) 40:201–211.[CrossRef][Web of Science][Medline]

3. Farmer H, McCabe N, Lord CJ, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature (2005) 434:917–921.[CrossRef][Medline]

4. Annab LA, Hawkins RE, Solomon G, et al. Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsive ovarian carcinoma cell line. Breast Cancer Res (2000) 2:139–148.[CrossRef][Web of Science][Medline]

5. Kamal GM, Gunaherath B, Leslie Gunatilaka AA, et al. 1,2(3)-Tetrahydro-3,30-biplumbagin: a naphthalenone and other constituents from Plumbago zeylanica. Phytochemistry (1983) 22:1245–1247.[CrossRef][Web of Science]

6. Alisa SM, Elazer RE. Glucose modulates basement membrane fibroblast growth factor-2 via alterations in endothelial cell permeability. J Biol Chem (2007) 282:14635–14644.[Abstract/Free Full Text]

7. Figtree GA, McDonald D, Watkins H, Channon KM. Truncated estrogen receptor alpha 46-kDa isoform in human endothelial cells: relationship to acute activation of nitric oxide synthase. Circulation (2003) 107:120–126.[Abstract/Free Full Text]

8. Osborne C, Wilson P, Tripathy D. Oncogenes and tumor suppressor genes in breast cancer: potential diagnostic and therapeutic applications. Oncologist (2004) 9:361–377.[Abstract/Free Full Text]

9. Ma YX, Tomita Y, Fan S, et al. Structural determinants of the BRCA1: estrogen receptor interaction. Oncogene (2005) 24:1831–1846.[CrossRef][Web of Science][Medline]

10. Xu J, Fan S, Rosen EM. Regulation of the estrogen-inducible gene expression profile by the breast cancer susceptibility gene BRCA1. Endocrinology (2005) 146:2031–2047.[Abstract/Free Full Text]

11. Fedier A, Steiner RA, Schwarz VA, et al. The effect of loss of Brca1 on the sensitivity to anticancer agents in p53-deficient cells. Int J Oncol (2003) 22:1169–1173.[Web of Science][Medline]

12. Tassone P, Tagliaferri P, Perricelli A, et al. BRCA1 expression modulates chemosensitivity of BRCA1-defective HCC1937 human breast cancer cells. Br J Cancer (2003) 88:1285–1291.[CrossRef][Web of Science][Medline]

13. Zhou C, Huang P, Liu J. The carboxyl-terminal of BRCA1 is required for subnuclear assembly of RAD51 after treatment with cisplatin but not ionizing radiation in human breast and ovarian cancer cells. Biochem Biophys Res Commun (2005) 336:952–960.[CrossRef][Web of Science][Medline]

14. Zhang GJ, Kimijima I, Onda M, et al. Tamoxifen-induced apoptosis in breast cancer cells relates to down-regulation of bcl-2, but not bax and bcl-X (L), without alteration of p53 protein levels. Clin Cancer Res (1999) 5:2971–2977.[Abstract/Free Full Text]

15. Feki A, Jefford CE, Berardi P, et al. BARD1 induces apoptosis by catalysing phosphorylation of p53 by DNA-damage response kinase. Oncogene (2005) 24:3726–3736.[CrossRef][Web of Science][Medline]

16. Hsu YL, Cho CY, Kuo PL, et al. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) induces apoptosis and cell cycle arrest in A549 cells through p53 accumulation via c-Jun NH2-terminal kinase-mediated phosphorylation at serine 15 in vitro and in vivo. J Pharmacol Exp Ther (2006) 318:484–494.[Abstract/Free Full Text]

17. Feki A, Jefford CE, Durand P, et al. BARD1 expression during spermatogenesis is associated with apoptosis and hormonally regulated. Biol Reprod (2004) 71:1614–1624.[Abstract/Free Full Text]

18. Flygare J, Falt S, Ottervald J, et al. Effects of HsRad51 overexpression on cell proliferation, cell cycle progression, and apoptosis. Exp Cell Res (2001) 268:61–69.[CrossRef][Web of Science][Medline]

19. Flouriot G, Brand H, Denger S, et al. Identification of a new isoform of the human estrogen receptor-alpha (hER-alpha) that is encoded by distinct transcripts and that is able to repress hER-alpha activation function 1. EMBO J (2000) 19:4688–4700.[CrossRef][Web of Science][Medline]

20. Kang HJ, Kim HJ, Kim SK, et al. BRCA1 modulates xenobiotic stress-inducible gene expression by interacting with ARNT in human breast cancer cells. J Biol Chem (2006) 281:14654–14662.[Abstract/Free Full Text]

21. Kang HJ, Kim HJ, Rih JK, et al. BRCA1 plays a role in the hypoxic response by regulating HIF-1alpha stability and by modulating vascular endothelial growth factor expression. J Biol Chem (2006) 281:13047–13056.[Abstract/Free Full Text]

22. Bae I, Fan S, Meng Q, et al. BRCA1 induces antioxidant gene expression and resistance to oxidative stress. Cancer Res (2004) 64:7893–7909.[Abstract/Free Full Text]

23. Fan S, Ma YX, Wang C, et al. p300 modulates the BRCA1 inhibition of estrogen receptor activity. Cancer Res (2002) 62:141–151.[Abstract/Free Full Text]

24. Sandur SK, Ichikawa H, Sethi G, et al. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) suppresses NF-kappaB activation and NF-kappaB-regulated gene products through modulation of p65 and IkappaB alpha kinase activation, leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents. J Biol Chem (2006) 281:17023–17033.[Abstract/Free Full Text]

25. Kawai H, Li H, Chun P, et al. Direct interaction between BRCA1 and the estrogen receptor regulates vascular endothelial growth factor (VEGF) transcription and secretion in breast cancer cells. Oncogene (2002) 21:7730–7739.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/4/696    most recent
mdm557v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Thasni, K. A.
Right arrow Articles by Priya, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thasni, K. A.
Right arrow Articles by Priya, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?