Levels Of Tumor Cell Apoptosis Research Articles

Intratumoral injection of mRNA encoding survivin in combination with STAT3 inhibitor stattic enhances antitumor effects

Intratumoral delivery of mRNA encoding immunostimulatory molecules can initiate a robust, global antitumor response with little side effects by enhancing local antigen presentation in the tumor and the tumor draining lymph node. Neoantigen-based mRNA nanovaccine can inhibit melanoma growth in mice by intratumoral injection. Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune responses by secreting immunosuppressive agents, such as reactive oxygen species (ROS). Suppression of STAT3 activity by stattic may reduce MDSC-mediated immunosuppression in the TME and promote the antitumor immune responses. In this study, in vitro transcribed mRNA encoding tumor antigen survivin was prepared and injected intratumorally in BALB/c mice bearing subcutaneous colon cancer tumors. In vivo studies demonstrated that intratumoral survivin mRNA therapy could induce antitumor T cell response and inhibit tumor growth of colon cancer. Depletion of CD8+ T cells could significantly inhibit survivin mRNA-induced antitumor effects. RT-qPCR and ELISA analysis indicated that survivin mRNA treatment led to increased expression of receptor activator nuclear factor-κB ligand (RANKL). In vitro experiment showed that MDSCs could be induced from mouse bone marrow cells by RANKL and RANKL-induced MDSCs could produce high level of ROS. STAT3 inhibitor stattic suppressed activation of STAT3 and NF-κB signals, thereby inhibiting expansion of RANKL-induced MDSCs. Combination therapy of survivin mRNA and stattic could significantly enhance antitumor T cell response, improve long-term survival and reduce immunosuppressive tumor microenvironment compared to each monotherapy. In addition, combined therapy resulted in a significantly reduced level of tumor cell proliferation and an obviously increased level of tumor cell apoptosis in CT26 colon cancer-bearing mice, which could be conducive to inhibit the tumor growth and lead to immune responses to released tumor-associated antigens. These studies explored intratumoral mRNA therapy and mRNA-based combined therapy to treat colon cancer and provide a new idea for cancer therapy.

Metadherin inhibits chemosensitivity of triple-negative breast cancer to paclitaxel via activation of AKT/GSK-3β signaling pathway.

Triple-negative breast cancer (TNBC) has an aggressive clinical course, and paclitaxel (PTX)-based chemotherapy remains the main therapeutic drug. Metadherin (MTDH) acts as an oncogene that regulates proliferation, invasion, metastasis, and chemoresistance. This study aimed to investigate whether TNBC chemosensitivity to PTX was related to the MTDH/AKT/glycogen synthase kinase-3beta (GSK-3β) pathway. Clinical baseline characteristics and immunohistochemistry (IHC) were used to evaluate the expression and prognosis of MTDH and AKT (protein kinase B, PKB) in TNBC patient samples. MTDH shRNA, MTDH overexpression vector, MK-2206, and PTX intervention were used in cell models and mouse tumor-bearing models. Afterwards, mRNA and protein levels were assessed using quantitative real-time polymerase chain reaction and Western blot. Evaluate the level of tumor cell apoptosis and cell cycle using flow cytometry. Cell viability was detected using Cell Count Kit 8. The invivo imaging system is used to analyze the growth of tumors. We found that higher expression of MTDH or AKT resulted in poorer disease-free survival and a lower Miller-Payne grade. MTDH promotes cell proliferation and increases p-AKT and p-GSK-3β expression in TNBC cells. Notably, suppression of AKT terminated MTDH overexpression-induced cell proliferation and apoptosis. MTDH knockdown or the AKT inhibitor MK2206 reduced the p-AKT and p-GSK-3β ratio, reduced cell viability and proliferation, increased cell apoptosis, and increased chemosensitivity to PTX. Invivo, xenograft tumors of an MTDH knockdown+MK2206 group treated with PTX were the smallest compared to other groups. In short, MTDH inhibits TNBC chemosensitivity to PTX by activating the AKT/GSK-3β signaling pathway.

LAMTOR1 degrades MHC-II via the endocytic in hepatocellular carcinoma.

Tumor cell surface antigen recognition is a major hallmark of cancer therapy, and loss of major histocompatibility complex class I (MHC-I) is the most common mechanism that impairs tumor cell surface antigen processing and expression. In addition to this, MHC-II regulates antigen presentation in CD4+ T cell immune responses involved in tumor killing by CD8+ T cells, whereas the regulation of endocytosis regulating MHC-II antigen presentation has not been reported. Therefore, the regulation of the endocytosis pathway on the expression of MHC-II surface level and antitumor T cell response remains to be explored. In this experiment, we found that LAMTOR1 regulates the endocytic pathway through the GTPase domain of DNM2 and triggers the formation of autophagosomes. We performed flow cytometry and western blotting analyses, which revealed that the expression of MHC-II molecules on the surface of cells is influenced by LAMTOR1 through the endocytic pathway. We showed that the expression of MHC-II molecules, which recognize CD4+ T cells on the surface of cells, was regulated by LAMTOR1 through an endocytic pathway. By coculture experiments, we showed that CD8+/CD4+ T cells exhibit substantially higher levels of tumor cell apoptosis than those observed when hepatocellular carcinoma (HCC) cells were cocultured with CD8+ T cells alone. This study revealed that LAMTOR1 decreases the expression levels of MHC-II on cell surfaces in order to reduce antigen expression, leading to a decrease in antitumor T cell responses.

Systemically Administered Reovirus-Induced Downregulation of Hypoxia Inducible Factor-1α in Subcutaneous Tumors

Reovirus, which possesses a 10-segmented double-stranded RNA genome, mediates superior antitumor effects via not only virus replication in a tumor cell-specific manner but also other mechanisms distinct from virus replication. Several groups, including ours, reported the reovirus-mediated downregulation of hypoxia inducible factor-1α (HIF-1α) following infection in cultured tumor cells; however, it remained to be clarified whether reovirus downregulates the expression of HIF-1α and its target genes in tumor-bearing hosts. We found that reovirus induced significant downregulation of protein levels of HIF-1α and its target genes in the subcutaneous tumors at 120 h post-systemic administration. Expression of reovirus capsid protein σ3 was found in the pimonidazole-positive hypoxic area in the tumor. Significant levels of tumor cell apoptosis were not found in the tumors of reovirus-treated mice at this time point, suggesting that reovirus-mediated tumor cell killing did not largely contribute to the downregulation of HIF-1α protein levels in the tumors. UV-inactivated reovirus did not induce downregulation of HIF-1α expression in the tumors, indicating that virus replication was indispensable for downregulation of HIF-1α expression in the subcutaneous tumors. This study provides important information for the development of reovirus-mediated virotherapy against various types of tumors.

Effects of peritumoral bevacizumab injection against oral squamous cell carcinoma in a nude mouse xenograft model: A preliminary study.

Angiogenesis serves a crucial role in tumor growth. Vascular endothelial growth factor (VEGF) is a potent regulator of tumor angiogenesis and is highly expressed in oral squamous cell carcinoma (OSCC). Bevacizumab, which binds to VEGF-A, inhibits the biological activity of VEGF and is clinically administered by intravenous injection. As intravenous chemotherapy intensifies the side effects experienced by OSCC patients, an alternative treatment option is desirable, particularly for older patients with OSCC who present with systemic disease complications. Generally, local injections of antitumor agents enhance tumoricidal activity and decrease side effects. However, the antitumor effects of peritumoral bevacizumab injections in OSCC are not fully understood. Therefore, the present study examined the effects of peritumoral bevacizumab injections in an experimental nude mouse model of OSCC through immunohistochemical staining for cluster of differentiation (CD)31 and α-smooth muscle actin (α-SMA) and apoptosis assays. It was identified that peritumoral injections of bevacizumab significantly inhibited tumor growth in OSCC xenografts compared with peritumoral saline injections or no treatment (controls), and it was also revealed that treatment with bevacizumab significantly reduced CD31- and α-SMA-positive microvessel density (P<0.01) and increased level of tumor cell apoptosis (P<0.01) compared with the controls. In conclusion, these results collectively support the experimental basis for the clinical development of peritumoral bevacizumab injections for the treatment of OSCC.

Antitumor effect and its mechanism analysis of chloroquine combination with chemotherapy for esophageal cancer in situ transplantation

Objective To explore antitumor effect and its mechanism analysis of Chloroquine combination with chemotherapy for esophageal cancer in situ transplantation. Methods 60 esophageal orthotopic transplantation tumor models were randomly divided into control group, 3-methyladenine (3-MA), paclitaxel and cisplatin combined therapy (TP) chemotherapy group and combination group. After 30 days treatment, tumor volume in four groups was analyzed. Autophagy substrate p62 and microtubule-associated protein 1 light chain 3 (LC3) were analyzed by Western blotting. VPS34 kinase activity was analyzed by kinase reaction. The apoptosis of tumor cells was determined by TdT-mediated dUTP nick end labeling (TUNEL) staining. The concentration of chemotherapeutic drugs in tumor cells of mice was analyzed by high performance liquid chromatography (HPLC) analysis. Results Compared with control group, tumor volume in TP chemotherapy group and combination group tumor growth significantly reduced (P=0.002). Compared with TP chemotherapy group, tumor growth in combination group of mice significantly reduced (P=0.010). Compared with the control group, p62 protein in tumor tissues in 3-MA and combination group significantly accumulated (P=0.000). Compared with control group, LC3-Ⅱ/β-actin ratio in 3-MA and combination group obviously decreased (P=0.000). Compared with the control group, VPS34 kinase activity in 3-MA and combination group obviously decreased (P=0.000). Compared with control group, tumor cell apoptosis levels in TP chemotherapy group and combination group significantly increased (P=0.000). Compared with TP chemotherapy group, the levels of tumor cell apoptosis in combination group significantly increased (P=0.000). Conclusion 3-MA can inhibit tumor cell autophagy, improve the utilization rate of drugs and enhance the anti-tumor effect. Key words: 3-Methyladenine; Paclitaxel and cisplatin combined therapy chemotherapy; Autophagy; Esophageal cancer; Drug resistance

Reduction stimuli-responsive unimolecular polymeric prodrug based on amphiphilic dextran-framework for antitumor drug delivery

We report a new reduction-responsive amphiphilic polymeric prodrug based on a linear dextran (DEX) backbone, which was conjugated with an hydrophobic camptothecin (CPT) prodrug block and an hydrophilic poly[poly(ethylene glycol) methyl ether methacrylate] (POEGMA) block [DEX-PCPT-b-POEGMA (DCO)] by atom transfer radical polymerization (ATRP). This amphiphilic prodrug has a unique molecular structure with prominent features, including strong practicability for methacrylate prodrug monomer, high drug loading rate (up to 23wt%), adjustable proportion of hydrophobic and hydrophobic portions, superior stability in aqueous solution, and easy access to cells. Introduction of a disulfide bond linker between the drug and the carrier can realize the function of reduction-responsive controlled drug-release. The experimental study indicated that the prodrug exhibited notable antitumor activity against HeLa cells and MCF-7 cells in vitro. Compared to similar DCO prodrug based on double carbon bond, the disulfide bond-conjugated DCO prodrug induced higher level of tumor cell apoptosis. Considering the drug-loading efficiency, micellar stability, cost of preparation and controlled drug release, the presented prodrug is more advantageous than traditional unimolecular prodrug and represents a promising approach for design of stimuli-responsive polymeric prodrug for effective cancer therapeutics.

Liver Injury and Tumor-Inhibiting Effect of Sequential Transcatheter Arterial Chemoembolization and Portal Venous Embolization on Rabbit VX2 Liver Carcinoma.

BackgroundThe concepts of sequential transcatheter arterial chemoembolization (TACE) and portal venous embolization (PVE) were proposed to prevent the detrimental tumor growth-inducing effect of PVE and to facilitate growth of further future liver remnant (FLR). This study aimed to investigate the effect of sequential TACE and PVE on liver damage and the therapeutic effect in a rabbit VX2 liver tumor model.Material/MethodsRabbits bearing VX2 liver tumors were randomly divided into TACE+PVE, TACE, PVE, and Sham groups. Plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), and alkaline phosphatase (ALP) at 6 h, 24 h, 3 days, and 7 days were measured by ELISA assay. Tumor diameter on day 7 was measured and the tumor sections with cleaved caspase-3 was stained and observed.ResultsPlasma ALT, AST, and ALP levels were significantly increased at the first hours after the interventions. The TACE group had higher increases than the TACE+PVE and PVE alone groups. ALT, AST, and ALP levels decreased on day 7 and presented a trend to return to the baseline level. The TACE+PVE group showed stronger tumor-inhibiting effect than the TACE and PVE alone groups and also induced the highest level of tumor cell apoptosis.ConclusionsThe liver damage caused by TACE+PVE is mild and recoverable. TACE+PVE showed stronger tumor-inhibiting effect than in the TACE and PVE group and also induced the highest level of tumor cell apoptosis.

Polymer–lipid hybrid nanoparticles synchronize pharmacokinetics of co-encapsulated doxorubicin–mitomycin C and enable their spatiotemporal co-delivery and local bioavailability in breast tumor

Effective combination chemotherapy requires the delivery of drugs of synergism to tumor sites while sparing normal tissues. Herein we investigated whether coencapsulation of doxorubicin and mitomycin C within polymer–lipid hybrid nanoparticles (DMPLN) achieved this goal via ratiometric drugs in an orthotopic murine breast tumor model with nanocarrier-modified biodistribution, pharmacokinetics, local bioavailability and toxicity. Fluorescence imaging revealed quickened and extended tumor uptake but reduced cardiac accumulation of DMPLN. Quantitative drug analysis demonstrated prolonged systemic circulation, increased tumor accumulation and sustained synergistic ratios of doxorubicin and mitomycin C delivered by DMPLN over 24h. Higher levels of tumor cell apoptosis and reduced organ toxicity were obtained with DMPLN compared to free drug cocktails. DMPLN released DOX in tumors more efficiently than that from liposomal doxorubicin, as evidenced by a higher extent of the metabolite, doxorubicinol. These findings substantiate the importance of rational design of nanoparticles for synergistic drug combination therapy. From the Clinical EditorThe treatment of cancer usually involves using combination chemotherapeutic agents. In adopting a nanomedicine approach, one can in theory design combination therapy consisting of drugs of synergistic activities, with the aim to target tumor specifically while minimizing systemic toxicity. The authors in this study provided evidence for this rational design by co-encapsulation of doxorubicin and mitomycin C within polymer-lipid hybrid nanoparticles (DMPLN) in a breast cancer model.

In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response.

A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo.

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.

Eradication of solid tumors using histone deacetylase inhibitors combined with immune-stimulating antibodies

Histone deacetylase inhibitors (HDACi) have been successfully used as monotherapies for the treatment of hematological malignancies; however, the single agent effects of HDACi against solid tumors are less robust. Using preclinical models of lymphoma, we have recently demonstrated that HDACi induce tumor cell-specific apoptosis and that this is essential for the therapeutic effects of these agents. Herein, we demonstrate that HDACi can be combined with immune-activating antibodies designed to promote the function of antigen-presenting cells (APCs) and enhance proliferation and survival of cytotoxic T cells (CTL) to stimulate a host antitumor immune response resulting in eradication of established solid tumors. This unique combination therapy was dependent on tumor cell apoptosis mediated by HDACi that stimulated the uptake of dead tumor cells by APCs. Tumor eradication was mediated by CD8(+) CTL that used perforin as the key immune effector molecule. This combination therapy was well tolerated and induced long-term immunological antitumor memory capable of mediating spontaneous tumor eradication upon rechallenge. These studies indicate that the ability of HDACi to mediate subtherapeutic levels of tumor cell apoptosis can be exploited by combining with antibodies that augment host antitumor immune responses to mediate robust and prolonged eradication of solid tumors.

Abstract 1411: A novel molecular approach for head and neck cancer radiosensitization

Abstract Purpose: Current combined external beam radiation therapy (XRT) and chemotherapy have demonstrated as an effective treatment against advanced head and neck cancers. However, combined modality treatments often result in unacceptable levels of toxicity to the patients. It has been well-known that enhanced DNA double strand break (DSB) repairing by Mre11, Rad50 and Nbs1 (MRN) complex is one of the most important mechanisms for developing radioresistance in the tumor cells. The present study investigates if the use of targeted Rad50 disruption could sensitize the tumor cells to XRT for the treatment of human head and neck squamous cell carcinoma (HNSCC). Our goal is to establish a most effective treatment strategy while minimizing XRT morbidity for the HNSCC. Method: Two human HNSCC tumor cell lines, JHU012 and JHU022, and an animal model with human HNSCC were used in this study. A dominant-negative recombinant adenoviral vector containing mutant Rad50 (Ad-Rad50) was constructed. The tumors were treated with Ad-Rad50, replication-defective virus (DL312), or untreated as mock control and a 2-Gy XRT. Cell viability and tumor volume were evaluated to analyze anti-tumor activity of different treatment groups. Neutral comet assay was performed for evaluating DNA DSB damage. Apoptosis in tumor cells was studied by using immunohistochemistry. Result: Our study demonstrates that the transgene expression of mutant Rad50 significantly interrupts the DNA DSB repairing and increases DNA damage in the tumor cells. Increased levels of tumor cell apoptosis were found associated with combined Ad-Rad50 and XRT treatment group. The combined Ad-Rad50 and XRT treatment shows the best anti-tumor result as compared to other groups both in vitro and in vivo. Conclusion: The present study suggests that Ad-Rad50 gene transfer effectively interrupts DNA DSB repairing and results in radiosensitizing in the HNSCC tumor cells treated with XRT. This strategy can be further investigated in the clinic for the treatment of advanced human head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1411.

Evaluation of tumor-infiltrating lymphocytes (TIL) and tumor cell apoptosis as predictive markers for response to neoadjuvant chemotherapy in triple-negative breast cancer

559 Background: Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor (ER), progesterone receptor (PgR) and HER-2. Pathological complete response (pCR) of TNBC to neoadjuvant chemotherapy (NAC) is correlated with excellent clinical outcome. We examined the value of histological parameters including tumor-infiltrating lymphocytes (TIL) and tumor cell apoptosis as surrogate markers for pCR in TNBC. Methods: Of 474 patients who received NAC and subsequent surgical therapy to stage II-III invasive breast carcinoma between 1999 and 2007, 102 (22%) had TNBC, and 92 core needle biopsy (CNB) specimens before NAC were available. We first immunohistochemically confirmed TNBC and basal-like subtype by current criteria for ER, PgR, and HER-2, cytokeratin (CK) 5/6, CK14, EGFR, and p53. All cases were TNBC, and 54 tumors (59%) were basal-like subtype defined as expression of at least one of CK5/6, CK14 and EGFR in &gt;1% of cancer cells. Totally, 26 tumors (28%) showed pCR. Thirteen histopathological parameters were examined, and their correlation with pCR rate was tested. These parameters were also examined in resected tumor specimens from 21 non-pCR cases. Results: The pCR rate was significantly higher in the patients with tumors with TIL (24 of 68, 35%) than in those without (2 of 24, 8%, p = 0.01), and higher in tumors with high-score apoptosis (9 of 19, 47%) than in those with low-score apoptosis (17 of 73, 23%, p = 0.04). Tumors showing medullary features and p53-negative tended to show pCR more frequently (38% and 35%) than those with non-medullary features and with p53-positive (25% and 24%), but the differences were not significant. Of 21 non-pCR cases, TIL was consistently negative before and after NAC in 8, but TIL emerged after NAC in 13. The pCR rate did not differ significantly between the basal-like type (31%) and non-basal-like type (24%). Conclusions: TIL and the level of tumor cell apoptosis appeared predictive markers for response to NAC in TNBC. Patients’ host factors correlated with immune response appears play a substantial role in the response to NAC in TNBC. No significant financial relationships to disclose.

Differential Effect of Doxorubicin and Zoledronic Acid on Intraosseous versus Extraosseous Breast Tumor Growth In vivo

Breast cancer patients with bone metastases are commonly treated with chemotherapeutic agents such as doxorubicin and zoledronic acid to control their bone disease. Sequential administration of doxorubicin followed by zoledronic acid has been shown to increase tumor cell apoptosis in vitro. We have therefore investigated the antitumor effects of clinically relevant doses of these drugs in a mouse model of breast cancer bone metastasis. MDA-MB-231/BO2 cells were injected via the tail vein into athymic mice. Tumor-induced osteolytic lesions were detected in all animals following X-ray analysis 18 days after tumor cell inoculation (day 18). Mice were administered saline, 100 microg/kg zoledronic acid, 2 mg/kg doxorubicin, doxorubicin and zoledronic acid simultaneously, or doxorubicin followed 24 h later by zoledronic acid. Doxorubicin-treated animals received a second injection on day 25. Tumor growth in the marrow cavity and on the outside surface of the bone was measured as well as tumor cell apoptosis and proliferation. The effects of treatments on bone were evaluated following X-ray and muCT analysis. Sequential treatment with doxorubicin followed by zoledronic acid caused decreased intraosseous tumor burden, which was accompanied by increased levels of tumor cell apoptosis and decreased levels of proliferation, whereas extraosseous parts of the same tumors were unaffected. Administration of zoledronic acid, alone or in combination with doxorubicin, resulted in significantly smaller tumor-induced osteolytic lesions compared with control or doxorubicin-treated animals. This is the first study to show that sequential treatment with clinically relevant doses of doxorubicin, followed 24 h later by zoledronic acid, reduces intraosseous but not extraosseous growth of BO2 breast tumors. Our results suggest that breast cancer patients with metastatic bone disease may benefit from sequential treatment using doxorubicin and zoledronic acid.

Cyclooxygenase 2 Expression in Rectal Cancer Is of Prognostic Significance in Patients Receiving Preoperative Radiotherapy

To determine the effect of cyclooxygenase (COX)-2 expression on clinical behavior in irradiated and nonirradiated rectal carcinomas. Tumor samples were collected from 1,231 patients of the Dutch TME trial, in which rectal cancer patients were treated with standardized surgery and randomized for preoperative short-term (5 x 5 Gy) radiotherapy or no preoperative radiotherapy. Tissue microarrays were constructed from primary tumor material, and COX-2 expression was assessed by immunohistochemistry. Tumor cell apoptosis was determined by M30 immunostaining. A high level of COX-2 expression after radiotherapy was associated with low levels of tumor cell apoptosis (P=0.001). COX-2 expression had no significant effect on patient survival or tumor recurrence in nonirradiated tumors. However, in patients receiving preoperative radiotherapy, high level of COX-2 expression was associated with higher incidence of distant recurrences [P=0.003; hazard ratio (HR), 1.7; 95% confidence interval (95% CI), 1.2-2.5] and shorter disease-free survival (P=0.002; HR, 1.8; 95% CI, 1.2-2.5) and overall survival (P=0.009; HR, 1.5; 95% CI, 1.1-2.0), independent of patient age, tumor stage, tumor location, or the presence of tumor cells in the circumferential resection margin. A high level of COX-2 expression after preoperative radiotherapy in resection specimens is associated with apoptosis resistance, high distant recurrence rates, and a poor prognosis in rectal cancer.

Longterm recombinant adeno-associated, virus-mediated, liver-generated expression of an angiogenesis inhibitor improves survival in mice with disseminated neuroblastoma.

A gene therapy-mediated approach to the delivery of antiangiogenic agents using adeno-associated virus (AAV) vectors has a number of advantages including the potential for sustained expression. The purpose of this study was to attempt to restrict the growth of disseminated neuroblastoma through delivery of a truncated, soluble form of the vascular endothelial growth factor receptor-2 (VEGFR-2 fetal liver kinase [Flk]-1), a decoy receptor for VEGF. Mice underwent portal vein injection of vector, either AAV-CAGG-tsFlk-1 or control vector. Subsequent truncated soluble fetal liver kinase-1 (tsFlk-1) expression was confirmed and quantified by ELISA. After 8 weeks, mice were given human neuroblastoma cells through the tail vein to establish disseminated disease and were sacrificed after 40 days. The weight of liver metastasis was measured. Intrahepatic tumor microvessel density and levels of apoptosis were determined. Survival was assessed in a second cohort of mice. After intraportal injection of AAV-CAGG-tsFlk-1, high-level, stable transgene expression was generated. Sera from these mice inhibited endothelial cell activation in vitro and Matrigel plug (BD Biosciences) neovascularization in vivo, suggesting that a systemic state of angiogenesis inhibition had been established. After neuroblastoma injection, mice expressing tsFlk-1 had a smaller tumor burden in the liver when sacrificed, as compared with control mice. The decrease in tumor weight in the liver correlated with lower intratumoral microvessel density and higher levels of tumor cell apoptosis in the tsFlk-1-treated tumors, supporting the hypothesis that decreased angiogenesis had occurred. In a second cohort of mice, survival of tsFlk-1-expressing mice after tumor cell challenge was longer than in control mice. Longterm in vivo expression of a functional VEGF inhibitor was established using an AAV-mediated gene therapy approach, and it demonstrated antiangiogenic and anticancer efficacy in a murine model of disseminated neuroblastoma.

T-Cell-Rich Large-B-Cell Lymphomas Contain Non-Activated CD8 + Cytolytic T Cells, Show Increased Tumor Cell Apoptosis, and Have Lower Bcl-2 Expression Than Diffuse Large-B-Cell Lymphomas

The factor(s) responsible for the reduced B cell number and increased T cell infiltrate in T-cell-rich large-B-cell lymphomas (TCRBCLs) have not been well characterized. We studied 18 TCRBCLs and 12 diffuse large-B-cell lymphomas (DLBCLs) to compare the 1) predominant T cell subpopulation(s), 2) expression of cytotoxic granule proteins (TIA-1 and granzyme B), 3) level of tumor cell apoptosis (Apoptag system, Oncor, Gaithersburg, MD), and 4) expression of Ki-67 (Mib-1) and apoptosis-related proteins (fas (CD95), bcl-2, and p53). T cells in TCRBCLs and DLBCLs were predominantly CD8+ T cells expressing alphabeta T-cell receptors and TIA-1 (16 of 18 TCRBCLs with >50% TIA-1+ small lymphocytes) but lacking granzyme B (16 of 18 TCRBCLs with <25% granzyme B+ small lymphocytes). Scattered apoptotic tumor cells (confirmed with CD20 co-labeling) were present in 15 of 18 TCRBCLs, with 14 of 15 cases having <10% apoptotic cells. No apoptotic cells were seen in 12 of 12 DLBCLs. In 16 of 16 immunoreactive TCRBCLs, <25% tumor cells were bcl-2+, whereas 6 of 12 DLBCLs had >50% bcl-2+ tumor cells. CD95 (fas) expression was also lower, with 3 of 18 (16.7%) TCRBCLs versus 4 of 12 (33%) DLBCLs having >25% CD95+ tumor cells. TCRBCLs and DLBCLs had similar levels of p53 and Ki-67 (Mib-1) expression. Thus, T cells in TCRBCLs are non-activated cytotoxic T lymphocytes (TIA-1+, granzyme B-). Tumor cell apoptosis (perhaps cytotoxic T cell mediated) may partly account for the decreased number of large (neoplastic) B cells in TCRBCLs, but other factors (ie, decreased bcl-2 expression) may also be needed.