Clusterin as a therapeutic target for radiation sensitization in a lung cancer model

PURPOSE: Clusterin plays important roles in cell survival and death. Inactivation of clusterin enhances the therapeutic efficacy of chemotherapy in lung cancer models. The purpose of this study was to determine whether inhibition of clusterin by an antisense-based investigative drug enhances radiation sensitization in a lung cancer model. METHODS AND MATERIALS: Cells were transfected with an antisense oligonucleotide (ASO) against clusterin (OGX-011). Apoptosis was determined by 7-aminoactinomycin D staining. Cell survival was examined by 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) and clonogenic assay. Xenograft model was used to demonstrate tumor growth and tumor blood flow. RESULTS: OGX-011 specifically attenuated the expression of secreted clusterin (prosurvival), with no apparent effect on the expression of nuclear clusterin (proapoptotic). Apoptosis was significantly increased when H460 lung cancer cells were treated with OGX-011 plus radiation. Inhibition of clusterin followed by radiation greatly decreased cell survival. H460 xenografts that were treated with OGX-011 plus radiotherapy demonstrated growth delay beyond 17 days. Doppler studies showed that tumor blood flow was compromised when mice bearing H460 xenografts were treated with OGX-011 and radiation. CONCLUSION: A combination of radiotherapy and OGX-011 improved control of tumor growth and vascular regression in the H460 lung cancer model.     

Synergistic antitumor effect of interferon-ß with gemcitabine in interferon-α-non-responsive pancreatic cancer cells

Interferon (IFN)-? is reported to have more potent antitumor effects than IFN-α. The aim of this study was to compare the synergistic antitumor activity of both IFNs when combined with gemcitabine on cultured pancreatic cancer cells expressing various levels of IFN receptor. The growth-inhibitory effects of IFN-α and IFN-? in combination with gemcitabine on three human pancreatic cancer cell lines (BxPC-3, MIAPaCa-2, Panc-1) were evaluated by MTT assay and isobolographic analysis. We also correlated their growth-inhibitory effects with the expression status of type I IFN receptor type 2 (IFNAR2). Western blot analysis indicated strong expression of IFNAR2 in BxPC-3 and MIAPaCa-2, but weak expression in Panc-1. The growth-inhibitory effect of gemcitabine was enhanced synergistically by IFN-α in BxPC-3 and MIAPaCa-2, but not in Panc-1. IFN-? exhibited more potent synergistic growth-inhibitory effects with gemcitabine in BxPC-3 and MIAPaCa-2 compared to IFN-α, and also synergistic enhancement in Panc-1. In conclusion, our results indicated that the growth-inhibitory effect of IFN-? with gemcitabine was synergistic not only in pancreatic cancer cells with strong expression of IFNAR2, but also in those with weak expression of IFNAR2.     

A phase I study of OGX-011, a 2'-methoxyethyl phosphorothioate antisense to clusterin, in combination with docetaxel in patients with advanced cancer.

PURPOSE: Clusterin is a cytoprotective chaperone protein that promotes cell survival and confers broad-spectrum treatment resistance. OGX-011 is a 2'-methoxyethyl-modified phosphorothioate antisense oligonucleotide that is complementary to clusterin mRNA, has a prolonged tissue half life, enhances drug efficacy in xenograft models, and reduces clusterin expression in humans with a biologically effective dose of 640 mg. The objective of this study was to determine a recommended phase II dose of OGX-011 in combination with docetaxel. EXPERIMENTAL DESIGN: Patients with cancers known from the literature to express clusterin were eligible. OGX-011 was given by 2-h i.v. infusion starting at 40 mg weekly after loading doses on days 1, 3, and 5. Docetaxel was given i.v. 30 mg/m(2) weekly for 5 of 6 weeks (schedule A) or 75 mg/m(2) every 3 weeks (schedule B). All patients had serial samples of peripheral blood mononuclear cells and serum assessed for clusterin expression. RESULTS: Forty patients were enrolled to eight cohorts. OGX-011 could be given at the full biologically effective single-agent dose of 640 mg with both docetaxel schedules. Toxic effects were primarily myelosuppression, fatigue, hair loss, gastrointestinal effects (expected docetaxel effects), as well as dose-related chills and fever (expected OGX-011 effects). OGX-011 AUC and C(max) increased proportionally with no apparent effect on docetaxel pharmacokinetics. At the end of cycle 1, serum clusterin showed mean decreases of 34% and 38% (range, 15-99%) at the 640-mg dose levels. CONCLUSIONS: OGX-011 can be given at a biologically effective dose with standard doses of docetaxel. Phase II trials of combined OGX-011 and chemotherapy are ongoing in patients with prostate, breast, and lung cancers.     

Clusterin inhibition using OGX-011 synergistically enhances antitumour activity of sorafenib in a human renal cell carcinoma model

Background:

The objective of this study was to investigate whether the therapeutic activity of sorafenib could be enhanced by combining with OGX-011, an antisense oligodeoxynucleotide (ODN) targeting clusterin, in renal cell carcinoma (RCC).

Methods:

We investigated the effects of combined treatment with OGX-011 and sorafenib on a human RCC ACHN model both in vitro and in vivo.

Results:

Although clusterin expression was increased by sorafenib, additional treatment of ACHN with OGX-011 significantly blocked the upregulation of clusterin induced by sorafenib. Despite the lack of a significant effect on the growth of ACHN, OGX-011 synergistically enhanced the sensitivity to sorafenib, reducing the IC₅₀ by >50%. Apoptotic changes were intensively detected in ACHN after combined treatment with OGX-011 and a sublethal dose of sorafenib, but not either agent alone. Furthermore, this combined treatment resulted in the marked downregulation of phosphorylated Akt and p44/42 mitogen-activated protein kinase in ACHN compared with treatment with either agent alone. In vivo systemic administration of OGX-011 plus sorafenib significantly decreased the ACHN tumour volume compared with control ODN plus sorafenib.

Conclusion:

Combined use with OGX-011 may be useful in enhancing the cytotoxic effect of sorafenib on RCC by inducing apoptosis and inactivating major signal transduction pathways.     

Treatment of pancreatic cancer xenografts with Erbitux (IMC-C225) anti-EGFR antibody,gemcitabine, and radiation

PURPOSE: To investigate treatment of human pancreatic cancer cell lines and xenografts with combinations of Erbitux (IMC-C225) anti-epidermal growth factor receptor (EGFR) antibody, gemcitabine, and radiation. METHODS AND MATERIALS: BxPC-3 and MiaPaCa-2 human pancreatic carcinoma cells were treated in vitro for 24 h with IMC-C225 (5 microg/mL), then exposed to epidermal growth factor (EGF) (10 mM) for 5 min. Immunoblots were screened for EGFR expression and the ability of IMC-C225 to block EGF-induced tyrosine phosphorylation of EGFR. Cells were treated with IMC-C225 (5 microg/mL) on Day 0, the IC(50) dose of gemcitabine on Day 1 for 24 h, followed by 3 Gy 60Co irradiation on Day 2, or the combination of each agent. For cell proliferation, cells were counted on Day 4, and for apoptosis, cells were stained with annexin V-FITC and propidium iodide, then analyzed by FACS. Cells were treated with the same single or multiple treatments and analyzed in a clonogenic cell survival assay. The effect of IMC-C225, gemcitabine, and radiation on the growth of BxPC-3 and MiaPaCa-2 tumor xenografts was determined. Athymic nude mice bearing established s.c. tumor xenografts of 6-8 mm diameter received 6 weeks of treatment with IMC-C225 (1 mg every 3 days x 6) alone or in combination with gemcitabine (120 mg/kg i.v. every 6 days x 6), and 6 weekly fractions of 3 Gy radiation on the days after gemcitabine administration. Tumor growth was measured with Vernier calipers. RESULTS: BxPC-3 and MiaPaCa-2 cell lines expressed low levels of EGFR. IMC-C225 inhibited EGF-induced tyrosine phosphorylation of the EGF receptor on both cell lines. Treatment of cells with a combination of IMC-C225 + gemcitabine + radiation produced the highest induction of apoptosis and inhibition of proliferation in vitro. Combination treatment with IMC-C225, gemcitabine, and radiation produced 100% complete regression of MiaPaCa-2 tumors for more than 250 days, and the greatest growth inhibition of BxPC-3 tumors compared to any single or dual treatments. CONCLUSIONS: The IMC-C225 therapy in combination with gemcitabine chemotherapy and radiation therapy demonstrated statistically significantly greater efficacy over the single and double combination therapies. This form of multimodality treatment shows potential clinical application in the treatment of pancreatic cancer in humans.     

A humanized anti-IGF-1R monoclonal antibody (R1507) and/or metformin enhance gemcitabine-induced apoptosis in pancreatic cancer cells

Pancreatic cancer is a disease with a dismal prognosis and treatment options are limited. This study investigated the interaction of gemcitabine with R1507 and/or metformin and the induction of an inhibitor of apoptosis protein by this com-bination. Pancreatic cancer cells were treated with gemcitabine, R1507 and metformin alone or in combination. The effects of treatments were evaluated for cell proliferation, apoptosis, and the expression of genes related to inhibition of apoptosis and chemotherapy resistance. Combination of gemcitabine with R1507 and/or metformin additively interacted with the inhibition of cell proliferation in human pancreatic ductal adenocarcinoma cell lines, SUIT-2 and MIAPaCa-2 with differential gemcitabine resistance, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index in both cell lines. Treatment with gemcitabine induced the expression of survivin and XIAP in both cell lines, indicating the induction of chemoresistance. In conclusion, these data demonstrate that the combination of gemcitabine with R1507 and/or metformin has an additive effect in pancreatic cancer cell lines with differential sensitivity to gemcitabine; however, gemcitabine may induce chemotherapy resistance.     

Clusterin is a potential molecular predictor for ovarian cancer patient's survival: targeting Clusterin improves response to paclitaxel

Background

Clusterin is a cytoprotective chaperone protein involved in numerous physiological processes, carcinogenesis, tumor growth and tissue remodelling. The purpose of this study was to investigate whether clusterin (CLU), an antiapoptotic molecule, could be a potential predictor molecule for ovarian cancer and whether or not targeting this molecule can improve survival of ovarian cancer patients.

Methods

Clusterin expression was compared between ten primary and their recurrent tumors from same patients immunohistochemically. We analyzed prognostic significance of CLU expression in another 47 ovarian cancer tissue samples by immunohistochemistry. We used small interference RNA to knock down CLU in the chemo-resistant ovarian cancer cell lines. KF-TX and SKOV-3-TX, paclitaxel-resistant ovarian cancer cells, were established from parental KF and SKOV-3 chemo-sensitive cell lines, respectively. Either siRNA or second generation antisense oligodeoxynucleotide against CLU (OGX-011), which is currently evaluated in clinical phase II trials in other cancer s, was used to modulate sensitivity to paclitaxel (TX) in ovarian cancer cells in vitro. Cellular viability assay, FACS analysis and annexin V staining were used to evaluate the comparative effect of CLU knocking down in ovarian cancer cells.

Results

Immunohistochemical analysis of CLU expression in primary ovarian cancer tissue specimens and their recurrent counterparts from same patients demonstrated higher expression of CLU in the recurrent resistant tumors compared with their primary tumors. High expression of CLU by immunohistochemistry among 47 surgical tissue specimens of early-stage (stage I/II) ovarian cancer, who underwent complete cytoreduction as a primary surgery, significantly related to poor survival, while none of other clinicopathological factors analyzed were related to survival in this patient cohort. Secretory CLU (s-CLU; 60 KDa) expression was upregulated in TX-resistant ovarian cancer cells compared to parental cells. Transfection of siRNA or OGX-011 clearly reduced CLU expression. Cell viability assay, FACS analysis and annexin V staining demonstrated that targeting CLU expression by siRNA or OGX-011 sensitized ovarian cancer cells to TX.

Conclusion

We conclude that CLU could be a potential molecular target to predict survival while targeting this s-CLU may improve survival of patients with ovarian cancer.     

Potentiation of the effect of erlotinib by genistein in pancreatic cancer: the role of Akt and nuclear factor-kappaB

The epidermal growth factor receptor (EGFR) is a target of new therapies in most nonhematologic cancers. EGFR blockade alone may not be sufficient for the control of growth and invasion of human pancreas cancer because of the independent activation of Akt and nuclear factor-kappaB (NF-kappaB). The expression of EGFR, Akt, and NF-kappaB was determined in six human pancreatic cancer cell lines. Selected cells for specific expression were treated with erlotinib, genistein, gemcitabine, or the combination. Growth inhibition was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was assayed by ELISA. EGFR, phosphorylated EGFR, phosphorylated Akt, and survivin expressions were determined by immunoblotting. Electrophoretic mobility shift assay was used to evaluate the DNA binding activity of NF-kappaB. Genistein significantly increased (P < 0.05) erlotinib-induced growth inhibition and apoptosis in BxPC-3, CAPAN-2, and AsPC-1 cells. In the BxPC-3 cell line, significant down-regulation of EGFR, phosphorylated Akt, NF-kappaB activation, and survivin was observed in the cells treated with the combination compared with the erlotinib-treated cells. In the HPAC and MIAPaCa cell line, no potentiation of the effects of erlotinib by genistein on cell growth or inhibition of the EGFR/Akt/NF-kappaB was observed. Genistein potentiated growth inhibition and apoptosis of the gemcitabine and erlotinib combination in COLO-357 cell line. Genistein potentiates the growth inhibition and apoptosis induced by erlotinib and gemcitabine in certain pancreatic cancer cells. Akt and NF-kappaB inhibition represents one of the mechanisms for the potentiation of erlotinib- and gemcitabine-induced cell death by genistein.     

RGD偶联吉西他滨白蛋白纳米粒对胰腺癌细胞周期和凋亡的影响

背景与目的:吉西他滨作为目前临床胰腺癌一线用药,由于其化疗耐药性,化疗效果较差。本研究通过制备RGD偶联吉西他滨白蛋白纳米粒,通过增加其靶向性,探讨其对胰腺癌细胞株BxPC-3和PANC-1细胞周期和凋亡的影响。方法:以人胰腺癌细胞株BxPC-3(高表达αvβ3受体)和PANC-1(低表达αvβ3受体)为研究对象,各分为6个给药组:对照组、BSANP(白蛋白纳米粒)、RGD-BSANP组、BSANP-GEM组、GEM原药组(吉西他滨)、RGD-BSANP-GEM组。运用PI单染检测给药后24 h细胞周期的变化,Annexin V/PI双染色法检测细胞凋亡。结果:在BxPC-3细胞株中,与BSANP-GEM组及GEM组相比,RGD-BSANP-GEM组S期细胞比例明显下降,G0/G1期细胞比例明显增多,且在RGD-BSANP-GEM组凋亡率最高;而在PANC-1细胞中,RGD-BSANP-GEM组G0/G1期细胞阻滞的比例和凋亡率低于BxPC-3细胞株。结论:RGD环肽能够增加BSANP-GEM对高表达αvβ3胰腺癌BxPC-3细胞G0/G1期阻滞及细胞凋亡。     

FR901228, a novel histone deacetylase inhibitor, induces cell cycle arrest and subsequent apoptosis in refractory human pancreatic cancer cells

Histone deacetylase (HDAC) inhibitors have antiproliferative activity against human cancer cells via cell cycle arrest, differentiation, and apoptosis. However, no report has focused on the apoptotic potential of HDAC inhibitors in refractory human pancreatic cancer. This study was designed to examine the apoptotic potential of FR901228, a novel HDAC inhibitor, in five human pancreatic cancer cell lines: Capan-1, BxPC-3, HPAF, Panc-1, and MIAPaCa-2. FR901228 markedly inhibited the proliferation of all five cell lines (IC50: 1-500 nM), with the greatest effect in MIAPaCa-2 cells. Treatment of each cell line with FR901228 (10-100 nM) caused cell cycle arrest at the G1 or G2/M phase and subsequent apoptosis. FR901228 induced expression of hyperacetylated histone H3 after 3 h of treatment and overexpression of p21Waf-1 after 6 h. In addition, FR901228 induced apoptosis by activating caspase-3, which led to cleavage of p21Waf-1 into a 15-kDa breakdown product and drove cancer cells from cell cycle arrest into apoptosis. FR901228 also decreased the protein level of survivin dramatically. Our results show that FR901228 markedly inhibits the growth of pancreatic cancer cells, not only through cell cycle arrest, but also through subsequent apoptosis; this was accompanied by caspase-3 activation, survivin degradation, and p21Waf-1 cleavage. FR901228 may prove clinically useful as an agent for refractory pancreatic cancers.     

Enhanced chemosensitivity of bladder cancer cells to cisplatin by suppression of clusterin in vitro

We examined the functional role of clusterin in chemotherapy-induced apoptosis and tested whether anti-sense transfection targeted against clusterin enhances the chemosensitivity in human bladder cancer cells in vitro. Clusterin mRNA and protein expression of 253J cells, a human bladder carcinoma cell line, after treatment with cisplatin were measured by RT-PCR and Western blot analysis. Clusterin expression and cell growth were compared between 253J cells transfected with constructed a clusterin anti-sense plasmid vector (pCR-CLU-AS) and controls. Tumor cell viability was measured with MTT assay after cisplatin treatment. DNA fragmentation and CPP32 assay were performed. Clusterin expression was increased after treatment with cisplatin and highest at 8 h in 253J cells. Clusterin anti-sense transfectants were highly sensitive to apoptotic cell death induced by cisplatin compared with parental 253J cells or control transfectants. Collectively, our results showed that expression of clusterin was increased in the acute phase of cell death caused by cisplatin and that suppressing the expression of clusterin enhanced the susceptibility of apoptosis caused by cisplatin in human bladder cancer cells. These results suggest that lowering the expression of clusterin might increase the sensitivity of bladder cancer cells to chemotherapeutic agents.     

Mechanisms underlying gemcitabine resistance in pancreatic cancer and sensitisation by the iMiD™ lenalidomide

Gemcitabine is currently the leading therapeutic for pancreatic cancer treatment, despite growing resistance. Studying the mechanisms that underlie gemcitabine resistance and discovery of agents that increase the tumour sensitivity to gemcitabine, is therefore desirable. The thalidomide analogue lenalidomide has been approved for use in multiple myeloma in combination with dexamethasone. Although it is primarily immunomodulatory, it also has direct effects on tumours. We investigated the sensitivity of three pancreatic cell lines PANC-1, MIA-PaCa-2 and BxPC-3 to gemcitabine. We observed that PANC-1 cells display most resistance to gemcitabine and MIA-PaCa-2 are most sensitive. Western blot analysis revealed that PANC-1 exhibits high phosphorylated extracellular signal-regulated kinase (pERK) expression, whereas MIA-PaCa-2 displays low expression. Combining gemcitabine and lenalidomide reduced the IC(50) of gemcitabine up to 40% (p<0.05). Western blot analysis showed lenalidomide significantly reduced pERK expression in all cell lines (p<0.05). It was hypothesised that gemcitabine sensitivity could be increased through combination with a pERK-reducing agent. The mitogen-activated kinase (MEK) specific inhibitor U0126 was used on PANC-1 cells to restore gemcitabine sensitivity. U0126 significantly increased cell killing by gemcitabine from 30% to 60% (p<0.001). Sensitive MIA-PaCa-2 cells were transfected with a constitutively active MEK mutant to reduce gemcitabine sensitivity. Transfection resulted in a significant reduction in cell killing by gemcitabine from 54-16% (p<0.05). These results provide evidence that ERK activity underlies sensitivity to gemcitabine and that addition of an agent that reduces this activity, such as lenalidomide, enhances gemcitabine efficacy. In conclusion, these results provide an understanding of gemcitabine resistance and could be used to predict successful combination therapies.     

Fluvastatin synergistically enhances the antiproliferative effect of gemcitabine in human pancreatic cancer MIAPaCa-2 cells

The new combination between the nucleoside analogue gemcitabine and the cholesterol-lowering drug fluvastatin was investigated in vitro and in vivo on the human pancreatic tumour cell line MIAPaCa-2. The present study demonstrates that fluvastatin inhibits proliferation, induces apoptosis in pancreatic cancer cells harbouring a p21ras mutation at codon 12 and synergistically potentiates the cytotoxic effect of gemcitabine. The pharmacologic activities of fluvastatin are prevented by administration of mevalonic acid, suggesting that the shown inhibition of geranyl-geranylation and farnesylation of cellular proteins, including p21rhoA and p21ras, plays a major role in its anticancer effect. Fluvastatin treatment also indirectly inhibits the phosphorylation of p42ERK2/mitogen-activated protein kinase, the cellular effector of ras and other signal transduction peptides. Moreover, fluvastatin administration significantly increases the expression of the deoxycytidine kinase, the enzyme required for the activation of gemcitabine, and simultaneously reduces the 5'-nucleotidase, responsible for deactivation of gemcitabine, suggesting a possible additional role of these enzymes in the enhanced cytotoxic activity of gemcitabine. Finally, a significant in vivo antitumour effect on MIAPaCa-2 xenografts was observed with the simultaneous combination of fluvastatin and gemcitabine, resulting in an almost complete suppression and a marked delay in relapse of tumour growth. In conclusion, the combination of fluvastatin and gemcitabine is an effective cytotoxic, proapoptotic treatment in vitro and in vivo against MIAPaCa-2 cells by a mechanism of action mediated, at least in part, by the inhibition of p21ras and rhoA prenylation. The obtained experimental findings might constitute the basis for a novel translational research in humans.     

IRF-2对胰腺癌细胞生物学特性及化疗敏感性的影响

目的 探讨干扰素调控因子2(interferon regulatory factor 2,IRF-2)在胰腺癌细胞中的生物学特性及其对吉西他滨化疗敏感性的影响.方法 Western blot检测IRF-2基因在胰腺癌细胞株PANC-1及MIAPaCa-2中的表达水平,采用MTT检测吉西他滨对PANC-1及MIAPaCa-2的半数有效浓度(IC50),选择较为耐药的PANC-1细胞进行IRF-2基因干扰表达,并采用MTT检测吉西他滨对PANC-1及干扰IRF-2表达的PANC-1 si1#的半数有效浓度(IC50).结果 PANC-1及MIAPaCa-2中细胞中均存在IRF-2基因的表达,且PANC-1细胞中表达水平比MIAPaCa-2高.吉西他滨对PANC-1细胞的IC50显著高于MIAPaCa-2细胞,差异有统计学意义(P＜0.05);干扰IRF-2表达的PANC-1 si1#细胞其IC50值显著低于PANC-1对照细胞,差异有统计学意义(P＜0.05).结论 IRF-2基因可作为影响胰腺癌对吉西他滨化疗敏感性的基因之一,干扰IRF-2基因能够有效地提升胰腺癌细胞对吉西他滨化疗的敏感性.