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Research Articles: Therapeutics, Targets, and Development
Hsp27 knockdown using nucleotide-based therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells
1 The Prostate Centre, Vancouver General Hospital and 2 Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada
Requests for reprints: Martin Gleave, The Prostate Centre, University of Columbia, Vancouver Hospital, 2660-Oak Street, Vancouver, BC, Canada V6H-3Z6. Phone: 604-875-5003; Fax: 604-875-5604. E-mail: m.gleave{at}ubc.ca
Abstract
Heat shock protein 27 (Hsp27) is a cytoprotective chaperone that is phosphoactivated during cell stress that prevents aggregation and/or regulate activity and degradation of certain client proteins. Recent evidence suggests that Hsp27 may be involved in tumor progression and the development of treatment resistance in various tumors, including bladder cancer. The purpose of this study was to examine, both in vitro and in vivo, the effects of overexpression of Hsp27 and, correspondingly, the down-regulation of Hsp27 using small interfering (si) RNA and OGX-427, a second-generation antisense oligonucleotide targeting Hsp27. Hsp27 overexpression increased UMUC-3 cell growth and resistance to paclitaxel. Both OGX-427 and Hsp27 siRNA decreased Hsp27 protein and mRNA levels by >90% in a dose- and sequence-specific manner in human bladder cancer UMUC-3 cells. OGX-427 or Hsp27 siRNA treatment induced apoptosis and enhanced sensitivity to paclitaxel in UMUC-3 cells. In vivo, OGX-427 significantly inhibited tumor growth in mice, enhanced sensitivity to paclitaxel, and induced significantly higher levels of apoptosis compared with xenografts treated with control oligonucleotides. Collectively, these findings suggest that Hsp27 knockdown with OGX-427 and combined therapy with paclitaxel could be a novel strategy to inhibit the progression of bladder cancer. [Mol Cancer Ther 2007;6(1):299–308]
Introduction
Bladder cancer is the second most common malignancy of the genitourinary tract and the fifth leading cause of cancer-related deaths of men in the western industrialized countries. Current treatments for superficial bladder cancer are of limited efficacy in preventing tumor recurrence and progression; after transurethral resection with or without intravesical chemotherapy, 50% to 70% of superficial bladder cancers will recur (1), and a significant proportion of patients with carcinoma in situ may develop invasive bladder cancer within 5 years (2). The prognosis of patients with invasive and/or metastatic bladder cancer is still extremely poor despite recent therapeutic advances (3). Cisplatin- and taxane-based combination chemotherapy is the mainstay of treatment for patients with advanced bladder cancer and is at least palliatively effective. However, the efficacy of combination chemotherapy is limited because of de novo drug resistance or the development of the cellular-resistant phenotype during treatment. Indeed, even in the era of intravesical therapy with Bacillus Calmette-Guerin and active systemic chemotherapy regimens, overall mortality rates for both bladder cancer and carcinoma in situ have remained constant in the U.S. (4). This underscores the need to identify mechanisms by which cancer cells inhibit the effects of chemotherapeutic agents, develop novel treatments, and consequently improve the survival of patients with advanced bladder cancer.
In addition to altered drug transport and metabolism, chemoresistance may also result from alterations in apoptotic "rheostat." Heat shock protein 27 (Hsp27) seems to play a crucial role in regulating the balance between cell death and cell survival, where its overexpression is associated with the suppression of apoptosis, increased cytoprotection, and resistance to treatment. Hsp27 belongs to a family of proteins that modulate a diverse range of homeostatic and pathogenic intracellular activities (5). Constitutively expressed Hsps are essential in cell homeostasis by acting as molecular chaperones to facilitate the transport, folding, and assembly of polypeptides. Hsp27 is phosphoactivated during cell stress to form oligomers that prevent aggregation and/or regulate activity and degradation of certain client proteins. Hsp27 expression is increased in a variety of malignancies, including prostate (6), breast (7), gastric (8), ovarian (9), and bladder cancers (10, 11). Furthermore, Hsp27 overexpression has been associated with multidrug resistance (12) and direct inhibition of apoptosis (13, 14) and is functionally linked to increased tumorigenicity and treatment resistance in breast (15) and colon (16) cancers.
Small molecule or antibody inhibitors that specifically target Hsp27 have not been reported. Antisense oligonucleotides (ASO) are powerful tools that specifically hybridize with complementary mRNA regions to form RNA/DNA duplexes to inhibit target gene expression in a sequence-specific manner. Several ASOs targeting genes involved in neoplastic progression have been evaluated as potential therapeutic agents (17, 18). In fact, the combined use of ASO with other compounds, such as chemotherapeutic agents, has shown synergistic antineoplastic effects in several tumor models (18, 19). OGX-427 (OncoGenex, Vancouver, BC, Canada) is a second-generation ASO, generated using the 2'-O-(2-methoxy) ethyl (2'-MOE) backbone, that targets Hsp27 mRNA. In this study, we functionally analyzed the role of Hsp27 in bladder cancer cell survival using the UMUC-3 cell line both in vitro and in vivo and identified a role for Hsp27 in promoting UMUC-3 cell growth and chemoresistance. We then tested the effect of OGX-427 on the growth of Hsp27-expressing human UMUC-3 bladder cancer cells and found that this drug candidate potently suppresses Hsp27 levels, decreases cell growth rates through the induction of apoptosis, and chemosensitizes UMUC-3 cells both in vitro and in vivo.
Materials and Methods
Cells and Reagents
UMUC-3 cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 10% fetal bovine serum. UMUC-3 cells are human transitional carcinoma cells that are high grade and highly invasive (20). Paclitaxel was purchased from Biolyse Pharma (St. Catherines, ON, Canada). Stock solutions of paclitaxel were prepared with PBS to the required concentrations before each in vitro experiment. Dr. Helen M. Burt (Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada) generously supplied polymeric micellar paclitaxel used for in vivo studies.
Lentiviral Infection of Hsp27 into UMUC-3 cells
Human Hsp27 cDNA was subcloned into the lentiviral vector pHR'-CMV-EGFP at the BamHI and XhoI sites. Two vectors were created for study: pHR'-CMV-Hsp27 and pHR'-CMV (empty vector). Clone identity was verified using restriction digestion analysis and plasmid DNA sequencing. Infectious lentivirus was generated by cotransfection of 1.5 x 106 293T cells with target plasmids with pCMV
R8.2 (carries the sequence necessary for viral assembly of lentivirus) and pMD.G that express the vesicular stomatitis virus envelop glycoprotein G(VSV-G) pseudotype as described previously (21, 22). The 293T cells were transfected with Hsp27-expressing, enhanced green fluorescent protein-expressing, or empty lentiviral vector (10 µg of each) using the calcium phosphate precipitation method (Promega Profection Mammalian Transfection Systems, Promega, Madison, WI). 293T cells were transfected for 12 to 15 h, after which fresh medium was added for 24 h. Following this, the virus-containing media were collected and filtered through a 0.45-µm filter. Early-passage UMUC-3 cells (passage 30) were plated in 10-cm plates, and competent retrovirus was added to 30 to 40 multiplicity of infection. The cells were passaged and harvested for fluorescence microscopy to verify green fluorescent protein expression. The whole cell lysates were collected to ensure the expression of Hsp27 by Western blotting. Images were captured using a Zeiss Axioplan II fluorescence microscope at x63 magnification followed by analysis with an imaging software (Northern Eclipse, Empix Imaging, Inc., Mississauga, ON, Canada). Analysis of focal localization was also done with Northern Eclipse and Adobe Photoshop 5.5 software.
OGX-427 and siRNA
2'-O-(2-methoxyethyl) ASO, OGX-427, was obtained from OncoGenex. The sequence of OGX-427 corresponds to the human Hsp27 translation initiation site (5'-GGGACGCGGCGCTCGGTCAT-3'). A mismatch oligodeoxynucleotide (ODN; 5'-CAGCAGCAGAGTATTTATCAT-3') was used as a control. The sequence of Hsp27 small interfering (si) RNA corresponds to the human Hsp27 site (5'-GUCUCAUCGGAUUUUGCAGC-3'; Dharmacon, Lafayette, CO). A scrambled siRNA (5'-CAGCGCUGACAACAGUUUCAU-3') was used as a control for RNA interference experiments.
Treatment of Cells with OGX-427 and siRNA
Cells were plated at the density of 4,000 cells per 1.9 cm2 and treated 1 day later for 1 or 2 days with ASO or siRNA, respectively. OligofectAMINE (Invitrogen-Life Technologies, Inc., Carlsbad, CA) was used as a transfection agent for both ODN or siRNA uptake into the cells. UMUC-3 cells were treated with various concentrations of ODN or siRNA after a preincubation for 20 min with 3 mg/mL OligofectAMINE in serum-free OPTI-MEM (Invitrogen-Life Technologies, Inc.).
Northern Blot Analysis
Total RNA was isolated from cultured UMUC-3 cells and tumor tissues using the TRIzol method (Invitrogen, Carlsbad, CA). Electrophoresis, hybridization, and washing conditions were carried out as reported previously (18, 23). Human Hsp27 cDNA probe was obtained from StressGen (Victoria, BC, Canada). The density of bands for Hsp27 was normalized against that of glyceraldehyde-3-phosphate dehydrogenase by densitometric analysis (Scion Image, Scion Corporation, Frederick, MD). Each assay was done in triplicate.
Western Blot Analysis
Samples containing equal amounts of protein (10-40 µg) from lysates of cultured UMUC-3 cells underwent electrophoresis on a SDS-polyacrylamide gel and were then transferred to a nitrocellulose filter. Filters were blocked in PBS containing 5% nonfat milk powder at 4°C overnight and then incubated for 1 h with a 1:5,000 diluted antihuman Hsp27 rabbit polyclonal antibody (StressGen), caspase-3 rabbit polyclonal antibody (New England BioLabs, Inc., Mississauga, ON, Canada), poly(ADP-ribose) polymerase rabbit polyclonal antibody (Cell Signaling Technology Inc., Beverly, MA) and antivinculin mouse monoclonal antibody (Sigma, St. Louis, MO). Filters were then incubated for 30 min with 1:5,000 diluted horseradish peroxidase–conjugate antirabbit or mouse monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Specific proteins were detected using an enhanced chemiluminescence Western blotting analysis system (Amersham Life Science, Arlington Heights, IL). The density of bands for Hsp27 was normalized against that of vinculin by densitometric analysis (Scion Image, Scion Corporation). Each assay was done in triplicate.
In vitro Cell Growth Assays
To examine the effect of Hsp27 overexpression on cell growth, 2 x 103 cells were seeded in 96-well microtiter plates, and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 24, 48, and 72 h as described previously (24). Briefly, the MTT assay is a quantitative method of determining the number of live cells to evaluate rates of cell growth or death. To assess chemosensitivity, cells were incubated in media plus paclitaxel at various concentrations for 48 h after 5 x 103 cells were plated. To determine the effect of down-regulation of Hsp27 in parental UMUC-3 cells, which constitutively express Hsp27, UMUC-3 cells were transfected with OGX-427 or control ODN. All cells were seeded 96-well microtiter plates. After 24 h, cells were then treated once daily with indicated concentration of ODN for 2 days. After 48 h, cell viability was measured by MTT assay.
To assess the effect of the combination treatment of OGX-427 plus paclitaxel, cells were transfected with 50 nmol/L OGX-427 or 50 nmol/L control ODN as described above and then treated with indicated concentrations of paclitaxel for 2 days. Cell viability was then determined by MTT assay.
Flow Cytometric Analysis
Flow cytometric analysis of propidium iodide–stained nuclei was done to quantify apoptotic rates using the sub-G0-G1 cell fraction, as described previously (18, 23). Briefly, UMUC-3 cells were plated and then transfected with ODN (with or without paclitaxel) as described above. The cells were trypsinized at 1 day after last treatment (with or without 25 nmol/L paclitaxel) and analyzed for relative DNA content on a dual laser flow cytometer (Beckman Coulter Epics Elite; Beckman, Inc., Miami, FL). Each assay was done in triplicate.
Assessment of Growth of UMUC-3 Tumor In vivo
Approximately 5 x 106 UMUC-3 cells were inoculated s.c. in the flank region of 8-week-old male athymic nude mice (Harlan Sprague-Dawley, Inc., Indianapolis, IN) via a 27-gauge needle under halothane anesthesia. Each experimental group consisted of 10 mice. When UMUC-3 tumors reached 100 mm3, mice were randomly selected for treatment with OGX-427 or control ODN alone. To assess the effects of OGX-427 monotherapy, 12 mg/kg OGX-427 or control ODN was injected i.p. once daily for 7 days followed by three weekly treatments thereafter. To assess whether OGX-427 enhances paclitaxel activity in vivo, when UMUC-3 tumors reached 500 mm3, mice were randomly selected for treatment with paclitaxel plus OGX-427 or control ODN. In combination treatment groups, 0.5 mg/kg paclitaxel was administered i.v. thrice per week from days 28 to 35 and from days 42 to 49. Tumor volume measurements were done once weekly and calculated by the formula length x width x depth x 0.5236 (25). Data points were expressed as average tumor volume levels ± SE. All animal procedures were done according to the guidelines of the Canadian Council on Animal Care and with appropriate institutional certification.
TUNEL Apoptosis Assay
For terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) assay, tissue sections (5 µm thick) of formalin-fixed, paraffin-embedded specimens were deparaffinized in xylene, rehydrated in graded alcohol, and transferred to PBS. The slides were rinsed twice with distilled water and treated with a 1:500 proteinase K solution (20 µg/mL) for 15 min, and endogenous peroxidase was blocked with 3% hydrogen peroxide in PBS for 12 min. The samples were washed thrice with distilled water and incubated for 10 min at room temperature with terminal deoxynucleotidyl transferase buffer. Excess terminal deoxynucleotidyl transferase buffer was drained, and the samples were incubated for 18 h at 4°C with terminal transferase and biotin-16-dUTP. The samples were then rinsed four times with TB buffer and incubated for 30 min at 37°C with a 1:400 dilution of peroxidase-conjugated streptavidin. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine (Invitrogen-Life Technologies, Inc.). The sections were then washed thrice with PBS, counterstained with Gill's hematoxylin (Biogenex, San Ramon, CA), and again washed thrice with PBS. The slides were mounted with a Universal Mount (Research Genetics).
Quantification of Cell Proliferation and Apoptosis
Effects of various treatments on tumor cell proliferation and apoptosis were determined by immunohistochemistry staining of tissue sections with anti–proliferating cell nuclear antigen antibodies and the TUNEL assay. The tissue was recorded using a cooled CCD Optotronics Tec 470 camera (Optotronics Engineering, Goleta, CA) linked to a computer and digital printer (Sony Corporation, Shinagawa, Tokyo, Japan). The density of proliferative cells and apoptotic cells was expressed as an average number of the five highest areas identified within a single 400x field.
Statistical Analysis
All results are expressed as the mean ± SD, and the significance of differences were by Student's t test or ANOVA-repeated measurement (SPSS 11.0 for Windows, Tokyo, Japan). P values <0.05 and <0.01 were considered significant by Student's t test and P < 0.01 by ANOVA-repeated measurement.
Results
Hsp27 Overexpression Increases Cell Growth and Chemoresistance in UMUC-3 Cells
Western blot analysis confirmed high expression levels of Hsp27 in Hsp27-transfected cells (UMUC-3-hsp27), compared with empty vector–transfected cells (UMUC-3-mock) and parental controls (UMUC-3; data not shown). Cell growth rates were higher in UMUC-3-hsp27 cells compared with parental cells and vector-only–transfected cells (P < 0.01). Moreover, Hsp27 overexpression increased resistance to paclitaxel; the IC50 of UMUC-3-Hsp27 cells was >50-fold higher than that in UMUC-3 or UMUC-3-mock cells (P < 0.01).
Sequence-Specific and Dose-Dependent Inhibition of Hsp27 Expression by OGX-427 and siRNA
OGX-427 significantly down-regulated Hsp27 in a dose-dependent fashion. As shown in Fig. 1A and B
, OGX-427 treatment significantly reduced Hsp27 mRNA levels by up to 80% in a dose-dependent manner, whereas Hsp27 protein levels, as shown in Fig. 1C and D, was reduced by >95% after OGX-427 treatment. Similarly, Hsp27 siRNA inhibited Hsp27 mRNA and protein levels in dose-dependent manner (Fig. 2A-D
).
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Xenografts treated with combination OGX-427 plus paclitaxel had significant growth inhibition compared with tumors treated with paclitaxel plus control ODN (Fig. 4D). These differences continued to increase after the second treatment of paclitaxel (day 49) until the end of the in vivo experiment. These results further illustrate the additive effects of combination therapy of OGX-427 and paclitaxel and corroborate the in vitro results.
To quantify the level of Hsp27 knockdown induced by OGX-427, xenografts treated with OGX-427 or control ODN, plus or minus paclitaxel, were analyzed by Northern blotting (Fig. 5A ). Hsp27 mRNA expression in OGX-427–treated tumors was significantly down-regulated compared with that of control ODN-treated tumors (Fig. 5B). TUNEL assay was done to quantify the difference in apoptotic cells in UMUC-3 tumor tissues. Xenografts treated with OGX-427 had significant higher amounts of apoptosis compared with tumors treated with control ODN (Fig. 5Ci and D).
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Alterations in the apoptotic rheostat of cancer cells contribute to the development of chemoresistance. Defining mechanisms of action of apoptotic-regulatory proteins associated with treatment resistance may identify strategies to selectively manipulate the sensitivity of cancer cells to therapy. Recent evidence suggests that Hsp27 plays an integral role in treatment resistance in many malignancies. Hsps are a family of highly conserved proteins in which expression is induced by cell stressors, such as hyperthermia, oxidative stress, and cytotoxic drugs (27). Hsps have attracted attention as new therapeutic targets for cancer, especially since the discovery and characterization of geldanamycin as an inhibitor of Hsp90 (28) and the targeting of clusterin (24, 29, 30) in which the product has Hsp-like function. Hsp27 is an ATP-independent chaperone that prevents the aggregation or regulate activity and degradation of certain client proteins.
Hsp27 interacts with key apoptosis-associated proteins and functions to inhibit cell death by a variety of mechanisms. Hsp27 prevents the formation of the apoptosome by either preventing the release of mitochondrial cytochrome c or directly sequestering cytochrome c in the cytosol after mitochondrial release (31). Hsp27 also directly interacts with and inhibits caspase-3 activation (26). Hsp27 can interfere with the extrinsic cell death pathway by inhibiting Daxx, a mediator of Fas-induced caspase-independent apoptosis (32). Furthermore, Hsp27 has been shown to inhibit apoptosis by decreasing the reactive oxygen species within cells by increasing glutathione and reducing the toxic effect of oxidized proteins (33). In addition, Hsp27 acts early during cell stress to stabilize and accelerate the recovery of actin filaments, thus preventing the disruption of the cytoskeleton (34). Hsp27 is also involved in the regulation of the serine/threonine kinase AKT (protein kinase B), an important signaling molecule for cell survival and proliferation downstream of growth factor stimulation (35). Hsp27 can exert its antiapoptotic effects through the enhancement of nuclear factor-
B activity, by facilitating proteasomal degradation of its main inhibitor I-B (36). Finally, we recently reported that Hsp27 interacts with and increases signal transducers and activators of transcription 3 levels and activity, a relevant transcription factor in many cancers, identifying yet another cytoprotective mechanism mediated by Hsp27 (37). Thus, Hsp27 is an attractive therapeutic target because its inhibition would affect multiple different pathways implicated in cancer cell survival and resistance as opposed to targeting a single pathway.
Hsp27 is highly expressed in many human tumors, including prostate, breast, ovarian, gastric, and bladder cancers (6, 8, 9, 38). Although Hsp27 is expressed at low levels in normal bladder epithelial cells (39), Hsp27 expression is induced in transitional cells after exposure to heat and carcinogens (40). Accordingly, Kassem et al. have shown, using an elegant cDNA microarray analysis with quantitative reverse transcription-PCR confirmation, that high levels of Hsp27 are associated with radioresistance, and more importantly, that low Hsp27 expression may be a marker to predict bladder tumors that are sensitive to radiotherapy (41). Furthermore, Lebret's analysis of 42 patients with superficial bladder cancer showed that Hsp27 was highly expressed (i.e., >50% of cells) in 83% of tumors (11). In our series of 11 carcinoma in situ specimens obtained from transurethral resection, a high level of Hsp27 was expressed in all specimens relative to normal controls (data not shown). However, to our knowledge, there have been no studies analyzing the functional significance of Hsp27 expression in bladder cancer.
In this study, we identify, for the first time, the functional role of Hsp27 in bladder cancer growth and treatment resistance. Hsp27 overexpression in UMUC-3 cells accelerates cell growth and increases resistance against paclitaxel, whereas Hsp27 knockdown by OGX-427 or Hsp27 siRNA in vitro enhances the induction of apoptosis and chemosensitizes cells to paclitaxel. In vivo, OGX-427 monotherapy significantly inhibited tumor growth, and the combination therapy of OGX-427 with paclitaxel significantly inhibited tumor growth and enhanced apoptosis compared with tumors treated with paclitaxel plus control ODN. Because UMUC-3 cells have mutant p53, OGX-427 likely induces p53-independent apoptotic triggers (42). Collectively, these findings show that Hsp27 helps mediate bladder cancer progression and development of treatment resistance to cytotoxic chemotherapy. The response to OGX-427 in UMUC-3 cells are similar to our previous reports of Hsp27 knockdown in LNCaP and PC-3 prostate cancer cell lines (18, 37). In these studies, OGX-427 enhanced the in vivo effects of castration-induced regression to delay androgen-independent LNCaP and PC-3 tumor growth and also enhances chemotherapy in vivo, emphasizing the additive role this ASO has in combination therapy regimes.
Most cases of locally advanced or metastatic bladder cancer initially respond to combination chemotherapy; however, the development of an acquired resistant phenotype frequently occurs with the progression of the disease (2). Recently, several investigators have shown that overexpression of antiapoptotic genes in bladder cancer cells, such as clusterin (43), Bcl-2 (44), or mutant-type p53 (45), helps mediate drug resistance through the inhibition of apoptosis induced by chemotherapeutic agents, suggesting that the approach of enhancing chemosensitivity by decreasing the expression of antiapoptotic genes may be a rational strategy for patients with advanced bladder cancer. Furthermore, recent studies provide preclinical proof of principle that targeting antiapoptotic genes using ASO can enhance apoptosis induced by conventional cytotoxic chemotherapy (46). Knockdown of Hsp27 using ASO like OGX-427 may also be a rational approach in the treatment of superficial bladder cancer, where the main goal is the prevention of tumor recurrence and progression to muscle-invasive disease. Unfortunately, current treatment regimens have limited success in achieving these goals. The accessibility of the bladder mucosa and the low systemic absorption through the bladder wall make intravesical therapy an excellent route of administration of ASO. Although no studies have yet been reported that assess pharmacokinetic and pharmacodynamic activity of ASO after intravesical delivery in humans, preclinical studies would suggest that ASO are taken up by urothelium (47, 48). Interestingly, the only approved clinical indication for an ASO is for intravitreal injection of cytomegalovirus-induced retinitis in human immunodeficiency virus, illustrating the potential clinical utility of local instillation for this class of therapeutics (49).
In summary, the present study suggests that the expression of Hsp27 helps mediate bladder cancer progression by inhibiting apoptotic cell death induced by cytotoxic chemotherapy. OGX-427 down-regulation of Hsp27 in UMUC-3 cells inhibits growth, induces apoptosis, and chemosensitizes cells to chemotherapy. These results provide preclinical proof of principle for the use of this novel therapeutic in the treatment of bladder cancer.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C Section 1734 solely to indicate this fact.
Received 7/18/06; revised 9/28/06; accepted 11/27/06.
References
B crystallin and Hsp27 in human urological tumor tissues. Urol Res 1998;26:395–9.[CrossRef][Medline] Lebret T, Watson RW, Molinie V, et al. Heat shock proteins Hsp27, Hsp60, Hsp70, and Hsp90: expression in bladder carcinoma. Cancer 2003;98:970–7.[CrossRef][Medline] Ciocca DR, Oesterreich S, Chamness GC, McGuire WL, Fuqua SA. Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review. J Natl Cancer Inst 1993;85:1558–70.B proteasomal degradation. Mol Cell Biol 2003;23:5790–802.
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100 mm3 (day 14), OGX-427 treatment was started. After 1 wk of an induction daily dose of 12 mg/kg/mouse OGX-427 or control ODN given i.p., mice were injected with the same dose thrice per week. Tumors were measured weekly. D, effects of combination therapy in vivo. Combination treatment was started when tumor volume reached 