Study Of The Effect Of Proteasome Inhibitor Bortezomib On Human Pancreatic Cancer Cells

1 Background and objectivePancreatic cancer is one of the most lethal gastrointestinal malignancies worldwide and surgical resection offers the only opportunity of cure currently. However, only 10%-20% of the patients are candidates for radical surgery because most patients present with locally advanced or metastatic disease at the time of diagnosis. Moreover, metastatic disease frequently develops even after potentially curative surgery. Accordingly, prognosis remains extremely poor, with 5-year survival rates of < 5%, and most patients with metastatic disease usually survive only 3-6 months. Systemic chemotherapy may increase survival and improve quality of life for patients with resected and locally advanced pancreatic cancer. Currently, gemcitabine has become the standard chemotherapeutic drugs used in pancreatic cancer. However, effectiveness of gemcitabine is lower than 20%.The ubiquitin-proteasome is essential for the disposal of damaged or unwanted proteins in eukaryotic cells. As a large multicatalytic protease complex, the proteasome is responsible for most non-lysosomal intracellular protein degradation. Through protein ubiquitination and degradation, the proteasome regulates many critical cell functions, such as cell cycle progression, gene transcription, and apoptosis. The role of the proteasome in regulating the growth and survival of tumor cells makes it an attractive therapeutic target. Bortezomib, a boronic acid dipeptide, is a specific and selective inhibitor of the proteasome currently under investigation as a potential anticancer therapy. In fact, Velcade (bortezomib) for injection is indicated for the treatment of multiple myeloma and mantle cell lymphoma in selected patients. Several studies on bortezomib's anticancer and chemosensitization effects in pancreatic cancer have been conducted, showing that it has significant activity in vitro and in vivo. However, the molecular mechanisms underlying proteasome inhibitor-mediated antitumor effects on pancreatic cancer are poorly understood. Therefore,understanding the changes occurring at the molecular level after gemcitabine and/or bortezomib treatment will allow a rational use of these agents and contribute to the design of novel combination regimens for pancreatic cancer patients.To gain insight into the molecular changes in treatments with bortezomib and gemcitabine, either alone or in combination, we examined global gene expression profiles of two pancreatic cancer cell lines using GeneChip Human Genome U133A 2.0 arrays.2 Materials and methodsGrowth Inhibition Assay Trypan blue exclusion assay was used to assess cell viability following the treatment with gradient concentration of bortezomib (1 nM, 10 nM, 100 nM, 1 uM, 10uM), and the concentration range of bortezomib for further experiment was determined. Cells were allowed to attach for 24 h and then effects of bortezomib and gemcitabine on growth of pancreatic cancer cells cultured for 24h, 48 h, 72h and 96h were examined using MTT assay.Tumor Growth and Treatments 1×10⁶ BxPC-3 cells collected in 100 μl serum-free DMEM media in log phase growth were injected subcutaneously in the backs of 20 g athymic nu/nu mice 4-6 weeks old. Once tumor masses became established and palpable (50mm³), animals were randomized to receive four weekly intraperitoneal (IP) injections of either vehicle (0.9% normal saline), bortezomib (1.0 mg/kg) alone, gemcitabine (125 mg/kg) alone, or bortezomib (1.0 mg/kg) and gemcitabine (125 mg/kg) in combination weekly for four treatments. Tumor volumes and body weight were measured weekly to evaluate antitumor effects of bortezomib in vivo.Apoptosis Cell cycle analysis and quantification of apoptosis analysis was carried out using propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS). Transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were also performed to study the apoptosis induced by bortezomib in vitro.Oligonucleotide Array Hybridization. Exponentially growing BxPC-3 and PANC-1 cells were treated with PBS or bortezomib. After 24 hour, gene expressionprofiles were determined using GeneChip HG U133A 2.0 arrays (Affymetrix, Santa Clara, California, USA) with 22278 probe-sets covering over 18 400 transcripts and variants representing 14 500 well-characterized human genes. After washing and staining, arrays were scanned at 570 nm using an Affymetrix GeneChip Scanner 3000 7G and analyzed using Affymetrics GeneChip Operating Software (GCOS). Gene ontology analysis was performed using the database for annotation, visualization, and integrated discovery (DAVID).Quantitative real-time reverse transcription- polymerase chain reaction (qRT-PCR) Selected differentially expressed genes were chosen to be validated by qRT-PCR using SYBR green I as fluorescent dye. The values of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used to normalize the expression data. Quantitation of gene expression was performed using the CT calculation, where CT is the threshold cycle. The gene expression level in various treated cells relative to the control cells was calculated using the following formulas: △△C_T= â–³C_Ttreated-â–³C_Tcontrol, fold change = 2^{△△CT}.Statistical Analysis Data were expressed as mean values ± SE and analyzed by a two-tailed t-test or ANOVA followed by the LSD's multiple comparison with P<0.05 considered significant. Analyses were performed using SPSS 11.5 statistical software package (SPSS Inc, Chicago, IL).3 ResultsEffects of Bortezomib on Growth of Pancreatic Cancer Cells For both cell lines, 80 nM bortezomib-induced cell growth inhibition was moderate at 24h (35.5% and 33% for BxPC-3 and PANC-1 respectively) and evident from 48h onwards (48.5% and 54% for BxPC-3 and PANC-1 respectively), while 160 nM was cytotoxic to almost all cells from 48h onwards for BxPC-3 and 72h onwards for PANC-1.Gemcitabine, on the other hand, displayed marked heterogeneity in responsiveness for two cell lines. After 48h and 72h of treatment, 0.05)μg/ml gemcitabine caused 48.33% and 76% decrease in BxPC-3 cell growth, while up to 50μg/ml of gemcitabine could induce similar results (45.67% and 67%) for PANC-1, demonstrating that BxPC-3was most and PANC-1 least sensitive to gemcitabine-induced growth inhibition.PANC-1 cells were treated with either 0.05-50 μg/ml gemcitabine alone or in combination with 40 nmol/L bortezomib. MTT assay at 48 h revealed that when cells were treated in combination, the decline in cell viability is more pronounced and reached statistical significance (P < 0.05) with larger dose of gemcitabine (50 μg/ml). As with low-dose gemcitabine treatment (0.05 μg/ml - 5 μg/ml), cells treated in combination displayed no significant growth inhibition, compared with gemcitabine alone. Similar results were seen in BxPC-3. Cells were treated with either 0.005-5 μg/ml gemcitabine alone or in combination with 40 nmol/L bortezomib for 48 hours. Bortezomib combination with gemcitabine (0.05 μg/ml - 5 μg/ml) caused significant decreased cells proliferation compared with gemcitabine alone (P < 0.01), and there was no statistical difference between treatment modality when dose of gemcitabine was 0.005 μg/ml.Effects of bortezomib and gemcitabine on subcutaneous xenograft tumors Either bortezomib or gemcitabine alone decreased tumor growth, and the combination of the two yielded a greater inhibition than either agent alone (P < 0.05). No mortality was observed and there was no difference in body weight among groups, indicating that system bortezomib alone or in combination with gemcitabine were well tolerated. Bortezomib Induces Apoptosis BxPC-3 and PANC-1 cells were treated with bortezomib, gemcitabine alone or its combination for 24 and 48 hours, and cell cycle analysis was performed as described. At either 24 or 48h, combined treatment resulted in a significant increase in the number of cells in the sub-G₀ phase, compared with gemcitabine alone, suggesting bortezomib is able to enhance the extent of gemcitabine-induced apoptosis.The effect of bortezomib on human pancreatic cancer cell lines was also examined by electron microscopy. In control BxPC-3 cells, the chromatin was floccular and dispersed throughout nuclei; the nuclear membrane was intact. In BxPC-3 cells treated with 100 nM bortezomib for 24 h, typical morphologic changes of apoptosis were seen. These early changes included chromatin condensation andmargination under the nuclear envelope. BxPC-3 cells in the later stage of apoptosis are shown after treated with 100 nM bortezomib for 48h, with more evident phenomenon of nuclear condensation and fragmentation. Similar results were observed in PANC-1 cells.Both gemcitabine and bortezomib as single agent induced a significant enhancement of apoptotic cells compared with control in pancreatic cancer cell lines BxPC-3. Apoptotic index (AI) was 7.298 ± 0.550% (gemcitabine) and 5.962 ± 0.426% (bortezomib) compared with 0.490 ± 0.010% of control (P<0.001). Maximum apoptotic effect was observed in the combination therapy group, which had a significantly higher number of TUNEL-positive cells (AI, 29.426± 1.223%) compared with the groups treated with either gemcitabine (P<0.001) or bortezomib alone (P<0.001). However, no significant differences were observed in AI between gemcitabine and bortezomib (P=0.198).Global gene expression profiles in pancreatic cancer cells treated by gemcitabine and bortezomib We profiled expression of probe sets using HG U133A 2.0 GeneChip and compared the mRNA expression of pancreatic cells treated with vehicle (PBS) and bortezomib (100 nM) for 24 h. In a comparison between various treatments and control groups and purging these sets of Affymetrix fragments for duplicate fragments from the same gene and ESTs, we identified 44 unique genes (52 fragments) up-regulated (>3-fold) and 24 unique genes (24 fragments) down-regulated (>5-fold) in both cell lines. Using DAVID (level 5 with a minimum of 4 gene), we found overexpression in a number of genes related to unfolded protein response (9 genes), cellular protein metabolism (20 genes) and apoptosis (8 genes). Further examination found Bcl-2 was repressed by bortezomib in both PANC-1 (2 fold) and BxPC-3 (3.73 fold) cells, while other members of Bcl-2 family such as Bax, Bak1, Bad, Bik and Bcl211 (Bcl-xl), etc, displayed no evident change. As for NF-kB family, unexpectedly, no changes induced in either cell by bortezomib were disclosed in all five members: NFKB1 (p105), NFKB2 (p100/p49), RelA (p65), RelB and Rel. Other interesting genes related to apoptosis or drug metabolism such as TP53 andABCB1 (mdr1) were not found differentially expressed in common.qRT-PCR verification of microarray data Five genes associated with apoptosis(GADD45B, DDIT3, PPP1R15A, HSPA1B and HSPB1) were chosen to verify themicroarray experiments. The change, observed with this technique, confirmed resultsobtained by the microarrays, although variations in magnitude of the change betweentwo technologies were observed.4 Conclusions1 Bortezomib inhibits growth of human pancreatic cancer cells in vitro and in vivo, and increases sensitivity to standard chemotherapeutic agent gemcitabine.2 Bortezomib induces apoptosis of human pancreatic cancer cells in vitro, and increases the effect of gemcitabine.3 Bortezomib exhibit pleiotropic effects, simultaneously up-regulating multiple cytotoxic and cytoprotective genes that function cooperatively in determining cell's fate.