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Intracellular Inhibitory Effects of Velcade Correlate with Morphoproteomic Expression of Phosphorylated-Nuclear Factor-B and p53 in Breast Cancer Cell Lines

Address correspondence to Robert E. Brown, M.D., Department of Laboratory Medicine, Geisinger Medical Center, 100 North Academy Ave., Danville, PA 17822, USA; tel 570 271 6333; fax 570 271 6105; e-mail rebrown{at}geisinger.edu.

	Abstract

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Velcade, a proteasome inhibitor, has been shown to inhibit DNA binding activity of nuclear factor-kappaB (NF-B) and to stabilize p53 in vitro. But its impact, in the context of activated (phosphorylated and translocated) NF-B and the expression of p53, has not been studied in breast cancer. It would be desirable to determine whether or not the immunohistochemical (IHC) expressions of activated NF-B and of p53 can predict the effects of Velcade in viable tumor cells. To answer these questions, we selected 3 breast cancer cell lines (SKBR-3, MDA-175, and MDA-231), which are negative for hormonal receptors, but differ in HER-2/neu expression (strong, mild, and minimal, respectively). The 3 cell lines showed different expressions of phosphorylated (p)- NF-B and p53, as evaluated using immunohistochemistry with visual quantification by brightfield microscopy. After being treated with Velcade for 2 days, MDA-231 cells showed markedly reduced proliferation, followed by SKBR-3 cells, and then by MDA-175 cells. There was strong correlation between the nuclear expression of either p-NF-B or p53 and the inhibitory rate of Velcade in the 3 cell lines (r = 0.987 and 0.807, respectively). Western blotting showed an increase in inhibitor-kappaB (I-B) expression in nuclei of MDA-231 and SKBR-3 cells, but not in MDA-175 cells, following exposure to Velcade. Velcade treatment resulted in cleaved caspase-3 expression in MDA-231 cells and in the overexpression of p53 and p21WAF1 in all 3 cell lines, as evaluated using Western blotting. In summary, morphoproteomic analysis of p-NF-B and p53 can be correlated with the inhibitory effect of Velcade in vitro. We propose that this proliferative inhibition is variably associated with blocking p-NF-B function by upregulation of nuclear I-B, stabilization of p53, and induction of p21WAF1.

(received 21 September 2004; accepted 30 September 2004)

Keywords: morphoproteomics, Velcade, proteasome inhibition, nuclear factor-kappaB, p53, apoptosis

	Introduction

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Velcade (bortezomib or PS-341), an inhibitor of the chymotryptic-like activity of the proteasome, has been shown to inhibit the proliferation of various types of tumors in vitro and in vivo [1–5]. Several studies report that Velcade treatment inhibits binding of nuclear factors-kappa B (NF-B) to DNA, possibly related to its inhibition of I-B degeneration in the proteasome [1,6]. In addition, Velcade treatment results in overexpression of p53 and p21WAF1 and activation of apoptosis [2,7,8]. Therefore, the mechanisms of Velcade in suppressing tumor cell growth appear to be multifactorial.

Because Velcade has antitumor activity against several human malignancies [9–11], it would be desirable to identify cellular makers to predict its inhibitory effects. Moreover, it would be ideal to stain malignant tumors immunohistochemically to assess predictable markers for guiding the selection of Velcade.

In this investigation, we examined the expression patterns of p-NF-B and p53 in association with apoptotic and growth inhibitory factors and the degree of growth inhibition in 3 human breast cancer cell lines following Velcade treatment. Our finding suggest that the immunohistochemical expression of p-NF-B and p53 is correlated with the in vitro growth inhibitory rate produced by Velcade and provides molecular concomitants to explain Velcade’s antitumoral effects in breast cancer cells.

	Materials and Methods

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Cells and materials.  Human breast cancer cell lines, SK-BR-3, MDA-(MB)-175, and MDA-(MB)-231 were obtained from American Type Culture Collection (ATCC) and were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM [Gibco-BRL,Gaithersburg, MD]), supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C in a 5% CO₂ atmosphere.

Velcade (bortezomib or PS-341) was obtained by from Millennium Pharmaceuticals, Inc. Cambridge, MA). It was prepared as a 2.6 mM stock solution in distilled water, and used at 10, 30, or 90 nM in cell culture medium.

Antibodies used in this investigation include rabbit polyclonal antibodies against I-B (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p-NF-B (p-p65 [Cell Signaling Technology, Inc., Beverly, MA]), cleaved caspase 3 (Cell Signaling), Bax (Cell Signaling), Bcl-2 (Cell Signaling), p53 (Santa Cruz), and p21WAF1/CIP1 (Santa Cruz).

Histologic techniques and immunohistochemistry.  Slides that each contained sections of 3 pelleted, formalin-fixed, and paraffin-embedded human breast carcinoma cell lines (SKBR-3,MDA-175, MDA-231) were obtained as part of the Hercept® kit (DAKO Corporation, Carpinteria,CA). These slides were stained immunohistochemically for p-NF-B (p-p65) and p53 with the respective antibodies (see Cells and Materials) and with the Vectastain ABC peroxidase kit (Vector Laboratories, Inc., Burlingame, CA). Immunoreactivities of the 3 cell lines were scored visually from 0 (negative) to 3+ positivity using brightfield microscopy. The final score for each protein analyte incorporated the range of signals and the relative percentages of positive cells within the individual cell line and reflected any heterogeneity among the tumor cells.

Cell proliferation assay.  SK-BR-3, MDA-(MB)-175, and MDA-(MB)-231 cells were plated in DMEM containing 10% fetal bovine serum (FBS) on 96-well culture dishes at 1x10⁴ cells/well. After 3 days of incubation, the medium was refreshed, and Velcade was added at the final concentrations indicated. Viable cells in each respective well were determined colorimetrically (CellTiter 96 one solution proliferation assay, Promega, Madison, WI) after 48 hr treatment. The cells were washed with phosphate-buffered saline (PBS), and then incubated for 1 hr at 37°C in modified Hank’s balanced buffer with the One Step Solution, which contains a tetrazolium compound. After the tetrazolium compound was bioreduced to a colored formazan, the absorbance was determined at 490 nm using a plate reader. The proliferation rates (viable cell numbers) were compared between the control and Velcade-treated cells, and proliferation inhibition rates were calculated ([control-treated]/control).

Western blotting.  At the end of treatment, cells were washed twice with ice-cold PBS and harvested in TN buffer for whole cell lysate, or in hypotonic buffer for nuclear extraction. Whole cell or nuclear homogenates, using 35 mg total protein per lane, were electrophoresed on 7–12% SDS PAGE. Fractionated proteins were transferred to nitrocellulose membranes. For immunostaining of p-NF-B, rabbit anti-p-NF-B antibody (1:1,000 dilution) was used. The second antibody was anti-rabbit horseradish peroxidase-linked whole antibody (1:3,000 dilution). Immunoreactive proteins were visualized by an enhanced chemiluminescence-Western blotting system (Amersham Phamacia Biotech, Piscataway, NJ). Western blots for other antibodies were performed as described above, using the respective primary and secondary antibodies.

Statistics.  Data are reported as mean ± SE. Statistical comparisons among the 3 groups were performed using ANOVA. Values of p <0.05 were considered to be significant.

	Results

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Correlation between the expression of p- NF-B and p53 and the effects of Velcade on cell proliferation.  The p-NF-B expression in the 3 human breast cancer cell lines (SKBR-3, MDA-175, and MDA-231), as analyzed by immunohistochemistry, revealed the highest level of p-NF-B in MDA-231, followed by SKBR-3, and the lowest in MDA-175 cells (Fig. 1A). Fig. 1B shows p53 immunoreactivity with the same expression pattern among the cell lines and notably with only mild (1+) nuclear p53 expression in the MDA-175 cell line.

View larger version (83K): [in this window] [in a new window]   Fig. 1. Immunohistochemical expression of p-NF-B and p53 in 3 breast cancer cell lines, which show the strongest overall signal in MDA-231, followed by SKBR-3, and then by MDA-175. Note the weakest (brown) immunoreactivity for intranuclear p53 in the MDA-175 cell line. A tumor cell, essentially nonimmunoreactive for p-NF-B antigen, has been included in each of the upper frames for comparison; (3-3'-diaminobenzidine tetrahydrochloride chromogen, magnification x 600).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Correlation of immunohistochemical (IHC) expression of nuclear p-NF-B and p53 with the inhibitory effect of Velcade in 3 breast cancer cell lines. Frame A gives a tabular summary of the visual scoring of nuclear immunoreactivity for p-NF-B and p53 on a scale of 0-3+, based on the consensus of two pathologists (PLZ and REB). The graph in Frame A shows the dose-response effect of Velcade and the growth inhibition (%) in the 3 cell lines. Frames B and C show the correlation between the nuclear staining scores for p-NF- B and p53 vs the inhibitory rate obtained with 30 nM Velcade (r = 0.987 and 0.807, respectively).

Velcade differentially modulates the levels of p53 and p21WAF1 in all 3 cell lines and activates the apoptotic caspase pathway in MDA-231 cells within a short period after exposure. Because the cell cycle checkpoint proteins, p53 and p21WAF1 are regulated, in part, by the ubiquitin-proteasome pathway [2,7,8,12–14],we studied the modulation of p53 and p21 WAF1 following Velcade treatment. Specifically, we examined the protein expression in whole cell and nuclear extracts after SKBR-3, MDA-175, and MDA-231 cells were exposed to 90 nM Velcade for 18 hr (Fig. 3). In MDA-175 cells, p53 was expressed at an undetectably low level (control), and Velcade treatment induced a detectable increase in p53 protein, mainly in nuclei. In SK-BR-3 cells and MDA-231 cells, Velcade treatment caused further accumulation of p53 in nuclei. Interestingly, no mater what status of p53 these cells have, Velcade increased p21WAF1 protein level expression in both whole cell and nuclear extracts in all 3 cell lines, especially, in the nuclei of MDA-231 cells.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Expression of p53, p21WAF1, and apoptotic factors following Velcade treatment for 18 hr (V), vs an untreated control (C) in each of the 3 cell lines. The Western blots show prominent expression of p53 in nuclei of MDA-231 and to a lesser extent SKBR-3, with undetectable nuclear p53 in MDA-175 controls (C); a discernible increase of nuclear p53 protein in all 3 cell lines following Velcade treatment (V); an increase of p21WAF1 protein level expression following Velcade (V) in the whole cell and nuclear extracts in all 3 cell lines, particularly in nuclei of MDA-231 cells; and essentially unchanged Bax and Bcl-xl levels, but increased cleaved caspase 3 expression in whole cell lysate in MDA-231 cells following Velcade (V). Note the actin bands that confirm equal loading of the control (C) and Velcade (V) lanes for each cell line.

Velcade causes accumulation of I-kB in nuclei of MDA-231 and SKBR-3 cells.  The regulated proteolysis of I-B is mediated by the ubiquitin-proteasome pathway[6,13]. Therefore, it is expected that Velcade treatment may cause accumulation of I-B in cells. To detect the expression of I- B, SKBR-3, MDA-175, and MDA-231 cells were treated with 90 nM Velcade for 6 or 18 hr, and whole cell lysate and nuclear extracts were collected and analyzed. Velcade did not induce accumulation of I-B in whole cell samples of MDA-231 and SKBR-3 cells, compared to the corresponding untreated control, but it caused increased I-B protein accumulation in nuclei of these cells in a time-dependent fashion (Fig. 4, upper panels). In Velcade-treated MDA-231 cells, the nuclear I-B level increased 2 and 3.7 times after 6 and 18hr, respectively, when analyzed using a computerized band-density measurement. In SKBR-3 cells, Velcade also caused increased nuclear I-B by 2.6 times at 18 hr. In contrast, there was no change in the I-B protein level in MDA-175 cells. After Velcade treatment, p-NF-B was overexpressed in all 3 cell lines (Fig. 4, lower panels). In MDA-231 and SKBR-3 cells, the nuclear accumulation of p-NF-B was proportional to the background levels. Velcade did not induce the accumulation of nuclear p-NF-B in MDA-175 cells.

View larger version (49K): [in this window] [in a new window]   Fig. 4. Expression of I-B and p-NF-B after Velcade treatment for 6 and 18 hr (V) versus untreated controls (C) in each of the 3 cell lines showing. The Western blots show increased I-B protein accumulation in nuclei of MDA-231 and SKBR-3 cells at 18 hr following Velcade (V), and overexpression of p-NF-B in whole cell lysates from all 3 cell lines following Velcade treatment (V) versus untreated controls (C) The overexpression is also evident in nuclear extracts from MDA-231 and SKBR-3, particularly the former. Note the actin bands that confirm equal loading of the control (C) and Velcade (V) lanes for each cell line.

	Discussion

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Since antibodies against p53 detect both wild type and mutated p53 in cancer cells and only the wild type of nuclear p53 is generally considered to be the functional form [16,17], the correlation in this instance, where p53 is the mutant type in both MDA-231 [18] and SKBR-3 [19], suggests possible activation of the mutated form. Evidence of such a mechanism has been proposed in other studies [2,20–23] and if confirmed for Velcade, adds to its attractiveness as a potential therapeutic agent. In short, the morphoproteomic approach may be useful to identify the protein expression level in tumor cells as one predictor of drug efficacy, before Velcade is used to treat individual patients.

When we examined I-B/NF-B and p53/ p21WAF1 signals together, in response to Velcade, the data showed that Velcade probably plays multiple roles in the inhibition of cell proliferation, mainly by causing I-B and p21WAF1 accumulation in nuclei. To expand on these, by binding to NF- B, I-B inhibits the translocation and transcriptional functions of NF-B. The classical signaling pathway leading to the activation of NF-B is mediated by I-kappaB kinase, resulting in phosphorylation and then degradation of I-B, in association with the ubiquitin-proteasome system[24]. Degradation of nuclear I-B is known to be the key factor permitting activation of NF-B and the degradation is regulated through nuclear proteasomes [25]. As previously noted, Velcade treatment had been shown to inhibit proteasome activity [1,6], which could account for the accumulation of I-B in the nucleus. In a study of multiple myeloma, Ma et al [6] showed an increase in p-I-B from 30 min to 5 hr after adding Velcade to the culture. However, there is no report on I-B expression at a later period of Velcade treatment.

We did not find any increase in I-B expression in whole cell samples, especially at 18 hr after adding Velcade to cell cultures, but we observed that I-B accumulated in the nuclei and the accumulation of nuclear I-B was proportional to background levels of p-NF-B (Fig. 4). Our results suggest that Velcade treatment may induce I-B translocation from the cytosol to the nucleus or that Velcade predominantly inhibits the proteasomes located in nuclei. It is possible that the higher the constitutive p-NF-B level that tumor cells have come to depend on, the greater the metabolic disturbance Velcade treatment might cause by promoting accumulation of nuclear I-B, to complex with and to inhibit the binding of p-NF-B to DNA. The latter could result in tumor cell cycle arrest and apoptosis, similar to that described in mantle cell lymphoma B cells [1].

In our study, nuclear expression of p53 was increased following Velcade, consistent with the findings by Williams and McConkey [2]. The balance between p53 as a pro-apoptotic factor and NF-B as an anti-apoptotic factor helps to determine the final cell fate after exposure to chemotherapeutic agents [26,27]. It appears that Velcade-induced p53 also might counter the effects of p-NF-B in nuclei (despite an elevated level of p-NF-B) by triggering the activation of pro-apoptotic factors leading to apoptosis[2]. In the current study, the reduced cell numbers in the MDA-231 cell line may be related, at least in part, to increased apoptosis, as suggested by the increased expression of cleaved caspase-3 following Velcade treatment [28]. Finally, Ling et al [3] reported increased reactive oxygen species (ROS) and mitochondrial membrane potential, following Velcade. Li and coworkers [29] showed that p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis. Therefore, during Velcade exposure, overexpressed p53 might contribute to ROS-associated apoptosis.

Williams and McConkey [2] found that Velcade does not promote phosphorylation on serines 15 and 20 of p53 and that p53 remains bound to its inhibitor, mdm2. Degradation of p53 by the 26S proteasome is mediated by the E3 ubiquitin ligase function of mdm2 [30,31]; it appears, therefore, that Velcade may not induce p53 expression by this proteasome pathway. Instead, Velcade is known to stimulate p53 translocation to the nucleus, enhance p53 DNA binding, accumulate p53-dependent transcripts, and activate a p53-responsive reporter gene via a novel mechanism [2]. As noted earlier, Velcade might even activate the mutated form of p53 [2]. The possibility that Velcade may induce p21WAF1 overexpression by a p-53-independent mechanism [32–34] cannot be excluded.

In summary, we have observed close correlation between the morphoproteomic expression of p-NF-B and nuclear p53 and the inhibitory effect of Velcade in breast cancer cell lines. Following Velcade treatment, nuclear accumulation of I-B, p53, and p21WAF1 were associated with reduction of the proliferation rate of the breast cancer cells. Our hope is that Velcade may have a future therapeutic role in selected cases of breast cancer that show specific biological characteristics, as noted in this work.

	Acknowledgments

 
Authors thank Ms. Ann O. Karosas and Dr. Jeffery W. Prichard for their assistance and Ms. Sharon Coup for her secretarial support.

	References

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