Effects of the proteasome inhibitor bortezomib on gene expression profiles of pancreatic cancer cells

BACKGROUND: Pancreatic cancer remains a highly chemoresistant malignancy. Gemcitabine is a widely used clinical chemotherapeutic agent against locally advanced and metastatic pancreatic cancer. Proteasome inhibitor bortezomib has been shown to result in enhanced cytotoxicity and apoptosis when used alone or in combination with gemcitabine in pancreatic cancer cell lines. MATERIALS AND METHODS: To determine the effect of bortezomib on gene expression profile of pancreatic adenocarcinoma cells with different sensitivity to gemcitabine, we used Affymetrix HG U133A 2.0 GeneChip (Santa Clara, CA) and measured changes induced by bortezomib in pancreatic cancer cell lines with high (BxPC-3) and low (PANC-1) sensitivity to gemcitabine, at time points 24 h. Selected genes were subsequently validated by quantitative real-time polymerase chain reaction. RESULTS: Forty-four common genes in both PANC-1 and BxPC-3 cells were identified as up-regulated (>3-fold) induced by bortezomib analyzed by microarray, which are associated with multiple cytotoxic and cytoprotective effects. Bcl-2 was repressed by bortezomib in both PANC-1 and BxPC-3 cells, while no changes induced in either cell by bortezomib were disclosed in all five members of nuclear factor-kappa B family. Other interesting genes related to apoptosis or drug metabolism, such as TP53 and ABCB1 (mdr1), were not found differentially expressed in common. CONCLUSIONS: Bortezomib exhibits antitumor effects toward pancreatic cancer in vitro and in vivo. Genes with divergent apoptotic effects are induced by bortezomib, which may become promising targets for pancreatic cancer treatment.     

Effects of bortezomib in sensitizing human prostate cancer cell lines to NK-mediated cytotoxicity

The proteasome inhibitor, bortezomib, has been demonstrated to sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Natural killer (NK) cells represent potent antitumor effector cells. They also express TRAIL. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing, and have the same effect in human prostate cancer cell lines (LNCaP and DU145). We found that bortezomib strongly inhibits proliferation in both cell lines. Furthermore, compared with LNCaP cells, DU145 cells are more sensitive to bortezomib-induced apoptosis. However, bortezomib is unable to sensitize these two cell lines to NK cell-mediated killing in short-term assays. In long-term assays, we found that killing mediated by activated NK cells following bortezomib treatment leads to greater antitumor effects than either treatment alone. In addition, treatment with bortezomib causes these cells to upregulate apoptosis-related mRNA as well as death receptors and downregulate the major histocompatibility class (MHC)-I molecule on the cell surface of DU145 cells. In contrast, LNCaP cells are not sensitized by this treatment. Death receptors and the MHC-I molecule did not change in this cell line. These data suggest that bortezomib can be used to sensitize prostate cancer cells to NK cell-mediated killing and improve current cancer therapies. This therapeutic strategy may be more effective in patients with androgen-insensitive prostate cancer.     

Schedule-dependent molecular effects of the proteasome inhibitor bortezomib and gemcitabine in pancreatic cancer

BACKGROUND: 26S proteasome inhibitors are a novel class of compounds entering clinical trials as a method to increase tumor sensitivity to standard chemotherapy. We determined the effect of alternate sequencing regimens of a proteasome inhibitor and gemcitabine on molecular and cellular responses in pancreatic cancer cells. MATERIALS AND METHODS: MIA-PaCa-2 human pancreatic cancer cells were treated with the proteasome inhibitor bortezomib either before, simultaneously or following exposure to gemcitabine. Expression of the cell cycle proteins p21(WAF1/CIP1) and p27(KIP1), and the anti-apoptotic protein BCL-2 were determined by Western blotting. Cell cycle changes and immediate or delayed induction of apoptosis were quantitated. RESULTS: Gemcitabine followed by bortezomib induced the greatest induction of apoptosis and long-term inhibition of cell growth. Bortezomib treatment led to accumulation of p21(WAF1/CIP1) and p27(KIP1) and decreased BCL-2; gemcitabine decreased p27(KIP1), induced BCL-2 and had no effect on p21(WAF1/CIP1). When these agents were given in combination or sequence, intermediate changes in these proteins were observed, and the alterations did not correlate with immediate or delayed induction of apoptosis. CONCLUSIONS: Inhibition of the 26S proteasome following chemotherapy appears to be the most effective regimen, though changes in BCL-2, p21(WAF1/CIP1), p27(KIP1) do not necessarily correlate with the cellular effects when various sequences are examined. Therefore, these proteins may not be the most appropriate surrogate markers of efficacy of this regimen. These data provide the background for the development of the optimal regimen to be used in clinical trials.     

A case of prostate cancer associated with osteolytic bone metastases]

It is well known that prostate cancer metastasizes bone with osteoblastic change and that osteolytic change is rare. We report a case of prostate cancer that had bone metastases which were all osteolytic. A 62-year-old man was referred to our department because of abnormal prostatic acid phosphatase and pain in his right upper arm. Digital examination revealed an enlarged and hard prostate. A computed tomographic scan revealed multiple osteolytic changes and a bone scintigraphy was positive at these sites. Histopathology of both prostate and humerus showed poorly differentiated adenocarcinoma. He received castration and antiandrogen as hormonal therapy, but the patient's prostate specific antigen did not normalize. Therefore this case was suspected to be hormone-refractory prostate cancer.     

Low cell motility induced by hsp27 overexpression decreases osteolytic bone metastases of human breast cancer cells in vivo.

The mechanisms controlling the formation of osteolytic bone metastases in patients with breast cancer are still poorly understood. To explore the role of motility in the establishment of osteolytic bone metastases, we have used a model of bone metastasis in which MDA-MB-231 breast cancer cells exhibiting low (hsp27-transfectants) and high (control-transfectant) endogenous cell motility were compared. We found that MDA-MB-231 cells exhibiting low cell motility were less capable of establishing osteolytic lesions. The number and the area of the osteolytic lesions in mice inoculated with low motility cells were both significantly smaller. Histomorphometry of bone lesions also demonstrated less tumor area in mice bearing hsp27 transfectants although there was no difference in the osteoclast number per square millimeter of tumor-bone interface. These data suggest that cell motility may be an important mechanism in the metastatic cascade of breast cancer cells to the bone and that controlling cell motility may be a useful target to prevent the establishment of osteolytic bone metastases.     

硼替佐米体外对前列腺癌细胞迁移和侵袭的影响及其机制

目的：探讨硼替佐米体外对前列腺癌细胞迁移和侵袭的影响及其机制。方法：选择对数生长的LNCAP前列腺癌细胞株,分别选择不同浓度的硼替佐米处理,在处理24h后进行前列腺癌细胞迁移和侵袭的检测,同时收集细胞进行炎症因子表达实验和Western blot检测分析。结果：LNCAP+硼替佐米(10nmol/L)组、LNCAP+硼替佐米(20nmol/L)组细胞的迁移指数与侵袭指数都明显低于对单独LNCAP组,并且IL-12及TNF-α表达量明显低于单独LNCAP组,对比差异都有统计学意义(P<0.05)不同浓度的硼替佐米处理LNCAP细胞24h后,FAK总蛋白无明显变化,对比差异都无统计学意义(P>0.05)。结论：替佐米能够有效的抑制前列腺癌LNCAP细胞的迁移和侵袭,其作用的发挥与抑制IL-12及TNF-α的表达分泌有关,但对FAK总蛋白的表达无影响。     

The proteasome inhibitor bortezomib inhibits intimal hyperplasia of autologous vein grafting in rat model

OBJECTIVE: Increasing evidence indicates that inflammation plays an important role in intimal hyperplasia (IH) induced by autologous vein grafts. The proteasome inhibitor bortezomib shows anti-inflammatory effects, so we used an autologous vein transplantation model to test whether bortezomib inhibits neointimal formation in transplant-induced vasculopathy. MATERIALS AND METHODS: We subjected 88 rats to autologous external jugular vein grafting surgery randomly assigned to be treated with bortezomib or vehicle. After 24 or 72 hours, rats were humanely killed and vein grafts processed for real-time RT-PCR (24 and 72 hours), ELISA (24 hours), or neutrophil chemotaxis assay (24 hours). Subsequently, rats were humanely killed at 1 and 2 weeks after grafting with samples processed for morphometric analysis. RESULTS: Bortezomib significantly inhibited IH at 2 weeks compared with untreated controls (P < .05). Expression of mRNA for vascular cell adhesion molecule-1, intercellular adhesion molecule-1, cytokine-induced neutrophil chemoattractant 2beta, monocyte chemoattractant-1, interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha markedly increased in injured vessels during the first day after surgery declining over the following 3 days. Bortezomib significantly attenuated gene expression and protein levels of most inflammatory mediators (P < .05), simultaneously inhibiting neutrophil chemotactic activity of vessel homogenates. CONCLUSIONS: Bortezomib inhibited neointimal formation at least partially by attenuating the inflammatory response in transplant-induced vasculopathy. It may become a novel vasoprotective agent in the clinical field.     

Carcinoma of the prostate with multiple osteolytic metastases simulating multiple myeloma. A case report

A patient with multiple osteolytic metastases--ribs, pelvis, spine, skull and mandible--from carcinoma of the prostate is described.     

Effects of the Bowman-Birk inhibitor on growth, invasion, and clonogenic survival of human prostate epithelial cells and prostate cancer cells

BACKGROUND: The Bowman-Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor with demonstrated anticarcinogenic activity in both in vitro and in vivo systems. METHODS: The effects of BBI and BBI Concentrate (BBIC), a soybean concentrate enriched in BBI, on cell growth, invasion, and/or survival were evaluated by the sulforhodamine B assay, a colony formation assay, the trypan blue dye exclusion assay and an in vitro invasion assay. The cells used in these studies were normal human prostate epithelial cells and prostate epithelial cell lines derived from embryonic prostate tissue (267B1) or benign prostatic hyperplasia (BPH) tissue (BRF-55T) and human prostate cancer cells established by Ki-ras oncogene transfection of 267B1 cells (267B1/Ki-ras) or from metastatic lesions of human prostate cancer (LNCaP and PC-3). RESULTS: BBIC had a statistically significant inhibitory effect on the growth and clonogenic survival of BRF-55T, 267B1/Ki-ras, LNCaP, and PC-3 cells. BBI also inhibited the growth of LNCaP cells and the clonogenic survival of BRF-55T and 267B1/Ki-ras cells and decreased the ability of LNCaP cells to invade across reconstituted basement membrane (Matrigel) when PC-3 cell-conditioned medium was utilized as the chemoattractant. BBI or BBIC did not affect the growth of normal prostate epithelial cells. CONCLUSION: BBI and/or BBIC could be a useful agent for treatment of prostate diseases.     

双氢睾酮对人前列腺癌细胞株PC-3细胞生长影响的体外研究

目的:探讨双氢睾酮(Dihydrotestosterone,DHT)对前列腺癌PC-3细胞株生长的影响。方法:采用MTT方法,利用不同浓度的DHT刺激非激素依赖性前列腺癌PC-3细胞株,在培养1、2、3、4天分别测定细胞活性。结果:在加用DHT后的第1天,10-8mol/L组细胞生长速度明显加快,MTTOD值与对照组比较差异有统计学意义(P<0.05)。在第4天,分别加用10-9mol/L、10-8mol/L、10-7mol/L、10-6mol/LDHT,MTTOD值与对照组比较差异均有统计学意义(P<0.05),10-5mol/LDHT组与对照组OD值比较差异无统计学意义(P>0.05)。结论:非激素依赖性前列腺癌PC-3细胞生长受雄激素的影响,低浓度DHT可促进PC-3细胞的生长。     

Testicular metastases from prostate cancer

Testicular metastases from prostate cancer is an uncommon occurrence and only 100 cases have been reported in the literature. Bilateral metastases are even more uncommon; however, testicular metatases may occur especially in cases of advanced prostate cancer. This article reports two cases with bilateral testicular metastases from prostate cancer.     

姜黄素对前列腺癌细胞核转录因子抑制蛋白表达的影响

目的观察姜黄素对前列腺癌细胞核转录因子抑制蛋白(IkBα)表达的影响,探讨姜黄素抑制前列腺癌细胞增殖的作用机制。方法分别用10、25、50、75和100μmol/L 浓度的姜黄素对雄激素依赖性及雄激素非依赖性前列腺癌细胞株 LNCaP 和 PC3进行干预,5、12和24 h 后采用噻唑蓝(MTT)比色法观察细胞增殖情况;采用流式细胞术测定24 h 后细胞周期变化;5 h 后 Western 印迹法检测细胞中 IkBα的表达。结果姜黄素显著抑制 LNCaP 及 PC3细胞的生长,呈剂量和时间依赖性;姜黄素将两种前列腺癌细胞阻滞于 G_2、M 期[LNCaP 与 PC3细胞,空白对照分别为(11.4±1.3)%与(17.3±1.7)%,100μmol/L 姜黄素作用后分别为(27.3±2.8)%与(33.4±4.0)%],从而诱导肿瘤细胞凋亡;姜黄素作用于 LNCaP 细胞后,细胞中 IkBα表达无变化(F=0.129,P>0.05);但作用于 PC3细胞后,细胞中 IkBα的表达明显增强,呈现出显著的剂量依赖性(F=31.618,P<0.05)。结论姜黄素通过活化 IkBα在 PC3细胞中的表达发挥抑制 PC3细胞增殖的作用。对于 LNCaP 细胞,姜黄素可能通过抗氧化、抑制细胞内代谢产物形成等方式抑制 LNCaP 细胞增殖。     

蛋白酶体抑制剂诱导肿瘤细胞凋亡研究进展

抗肿瘤药物治疗主要面临抗癌效果和毒副反应这一矛盾,同时还出现耐药的肿瘤细胞株,使得肿瘤治疗难有较大进展.蛋白酶体抑制剂是新发现的具有抗癌效果的药物,它通过抑制泛素-蛋白酶体系统在细胞内的生物活性来调节细胞周期,促进细胞凋亡而起到抗癌作用,并可与其他化疗药物协同作用,减少耐药性.体内外实验研究发现蛋白酶体抑制剂对包括骨肉瘤在内的多种实体肿瘤细胞具有促凋亡的生物活性,可通过多种信号转导途径引起细胞凋亡.蛋白酶体抑制剂bortezomib已通过美国FDA认证而应用于临床治疗多种肿瘤并取得较好疗效.该文就蛋白酶体结构、蛋白酶体抑制剂作用途径、促凋亡作用机制的研究成果和面临问题、bortezomib临床应用前景作一综述.     

Differences in the cytokine profiles associated with prostate cancer cell induced osteoblastic and osteolytic lesions in bone.

Prostate adenocarcinoma is associated with the formation of osteoblastic metastases in bone. It is hypothesized that osteoclastogenesis is a critical component in the development of skeletal metastases. These findings, however, were generally noted in predominantly osteolytic lesions. The pathophysiology of osteoblastic lesions remains unknown but the type of bone lesion formed may be influenced by the cytokines produced by prostate tumors. To test this theory, we implanted PC-3 and LAPC-9 cells into the tibias of SCID mice. These mice were sacrificed at 1, 2, 4, 6, and 8 weeks after implantation and histologic analysis was performed on these tibias. PCR analysis was also performed on bulk tumors. The results showed that the PC-3 implanted tibias developed pure osteolytic lesions while the LAPC-9 implanted tibias developed pure osteoblastic lesions on radiographs. Analysis of tibias after injection with PC-3 cells revealed progressive osteolytic lesions with abundant osteoclast activity at 2 weeks and destruction of the proximal tibia at 6 weeks after cell implantation. In contrast, the LAPC-9 cells formed osteoblastic lesions six weeks after cell injection. There were rare osteoclasts prior to the establishment of the osteoblastic lesions but greater osteoclast activity was noted with remodeling of the osteoblastic lesion 8 weeks after implantation of the tumor cells. PCR analysis revealed that PC-3 cells produced RANKL, IL-1, and TNF-alpha, which are associated with osteoclastogenesis. In contrast, LAPC-9 cells produced osteoprotegerin, which blocks osteoclast production and no detectable levels of RANKL or IL-1 and only minimal amounts of TNF-alpha were noted. These cells secreted BMP-2, -4, -6, and IL-6, which are associated with bone formation. These results suggest that the role of the osteoclast in the development of a metastatic lesion is variable depending on the phenotype of the prostate cancer cells, and that tumor-induced osteolysis may not be required for osteoblastic metastases.     

Chromosome 18 suppresses prostate cancer metastases

Loss of heterozygosity and allelic imbalance data has shown that there are two distinct regions of loss on chromosome 18q associated with the progression of prostate cancer (CaP). To investigate the functional significance of chromosome 18q loci in CaP, we utilized the technique of microcell-mediated chromosome transfer to introduce an intact chromosome 18 into the human prostate cancer cell line, PC-3. Three of the resulting hybrid lines were compared to the PC-3 cells in vitro and in vivo. The hybrid cell lines, containing an intact copy of the introduced chromosome 18, exhibited a substantial reduction in anchorage-dependent and independent growth in vitro. These hybrid cell lines also made smaller tumors in nude mice following subcutaneous injection compared to PC-3 cells. Because tumor growth was not completely eliminated by introduction of chromosome 18, we assessed the ability of the hybrids to metastasize to bone after intra-cardiac inoculation in a nude mouse model. Mice inoculated with PC-3 hybrids containing intact copies of chromosome 18 had significantly fewer bone metastases and dramatically improved survival compared to PC-3 cells. In addition, the introduction of chromosome 18 significantly reduced tumor burden in extraskeletal sites. This was not because of differences in growth rates because mice bearing hybrids were monitored for metastases over twice as long as mice bearing PC-3 cells. Taken together, these data suggest that chromosome 18 has a functional role in CaP to suppress growth and metastases. Identification of the responsible gene(s) may lead to molecular targets for drug discovery.