Arsenic Trioxide as a Vascular Disrupting Agent: Synergistic Effect with Irinotecan on Tumor Growth Delay in a CT26 Allograft Model

The mechanism of action of arsenic trioxide (ATO) has been shown to be complex, influencing numerous signal transduction pathways and resulting in a vast range of cellular effects. Among these mechanisms of action, ATO has been shown to cause acute vascular shutdown and massive tumor necrosis in a murine solid tumor model like vascular disrupting agent (VDA). However, relatively little is understood about this VDA-like property and its potential utility in developing clinical regimens. We focused on this VDA-like action of ATO. On the basis of the endothelial cell cytotoxicity assay and tubulin polymerization assay, we observed that higher concentrations and longer treatment with ATO reduced the level of α- and β-tubulin and inhibited the polymerization of tubulin. The antitumor action and quantitative tumor perfusion studies were carried out with locally advanced murine CT26 colon carcinoma grown in female BALB/c mice. A single injection of ATO intraperitoneally displayed central necrosis of the tumor tissue by 24 hours. T1-weighted dynamic contrast-enhanced magnetic resonance image revealed a significant decrease in tumor enhancement in the ATO-treated group. Similar to other VDAs, ATO treatment alone did not delay the progression of tumor growth; however, ATO treatment after injection of other cytotoxic agent (irinotecan) showed significant additive antitumor effect compared to control and irinotecan alone therapy. In summary, our data demonstrated that ATO acts as a VDA by means of microtubule depolymerization. It exhibits significant vascular shutdown activity in CT26 allograft model and enhances antitumor activity when used in combination with another cytotoxic chemotherapeutic agent.     

KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway

We investigated the effects of KML001 (NaAsO₂, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-_L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-_L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice.     

Dll4 Blockade Potentiates the Anti-Tumor Effects of VEGF Inhibition in Renal Cell Carcinoma Patient-Derived Xenografts

Background

The Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis. Blockade of the Dll4-Notch pathway in preclinical cancer models has been associated with non-productive angiogenesis and reduced tumor growth. Given the cross-talk between the vascular endothelial growth factor (VEGF) and Delta-Notch pathways in tumor angiogenesis, we examined the activity of a function-blocking Dll4 antibody, REGN1035, alone and in combination with anti-VEGF therapy in renal cell carcinoma (RCC).

Methods and Results

Severe combined immunodeficiency (SCID) mice bearing patient-derived clear cell RCC xenografts were treated with REGN1035 and in combination with the multi-targeted tyrosine kinase inhibitor sunitinib or the VEGF blocker ziv-aflibercept. Immunohistochemical and immunofluorescent analyses were carried out, as well as magnetic resonance imaging (MRI) examinations pre and 24 hours and 2 weeks post treatment. Single agent treatment with REGN1035 resulted in significant tumor growth inhibition (36–62%) that was equivalent to or exceeded the single agent anti-tumor activity of the VEGF pathway inhibitors sunitinib (38–54%) and ziv-aflibercept (46%). Importantly, combination treatments with REGN1035 plus VEGF inhibitors resulted in enhanced anti-tumor effects (72–80% growth inhibition), including some tumor regression. Magnetic resonance imaging showed a marked decrease in tumor perfusion in all treatment groups. Interestingly, anti-tumor efficacy of the combination of REGN1035 and ziv-aflibercept was also observed in a sunitinib resistant ccRCC model.

Conclusions

Overall, these findings demonstrate the potent anti-tumor activity of Dll4 blockade in RCC patient-derived tumors and a combination benefit for the simultaneous targeting of the Dll4 and VEGF signaling pathways, highlighting the therapeutic potential of this treatment modality in RCC.     

Metronomic chemotherapy with low-dose cyclophosphamide plus gemcitabine can induce anti-tumor T cell immunity in vivo

Several chemotherapeutic drugs have immune-modulating effects. For example, cyclophosphamide (CP) and gemcitabine (GEM) diminish immunosuppression by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), respectively. Here, we show that intermittent (metronomic) chemotherapy with low-dose CP plus GEM can induce anti-tumor T cell immunity in CT26 colon carcinoma-bearing mice. Although no significant growth suppression was observed by injections of CP (100 mg/kg) at 8-day intervals or those of CP (50 mg/kg) at 4-day intervals, CP injection (100 mg/kg) increased the frequency of tumor peptide-specific T lymphocytes in draining lymph nodes, which was abolished by two injections of CP (50 mg/kg) at a 4-day interval. Alternatively, injection of GEM (50 mg/kg) was superior to that of GEM (100 mg/kg) in suppressing tumor growth in vivo, despite the smaller dose. When CT26-bearing mice were treated with low-dose (50 mg/kg) CP plus (50 mg/kg) GEM at 8-day intervals, tumor growth was suppressed without impairing T cell function; the effect was mainly T cell dependent. The metronomic combination chemotherapy cured one-third of CT26-bearing mice that acquired tumor-specific T cell immunity. The combination therapy decreased Foxp3 and arginase-1 mRNA levels but increased IFN-γ mRNA expression in tumor tissues. The percentages of tumor-infiltrating CD45⁺ cells, especially Gr-1^high CD11b⁺ MDSCs, were decreased. These results indicate that metronomic chemotherapy with low-dose CP plus GEM is a promising protocol to mitigate totally Treg- and MDSC-mediated immunosuppression and elicit anti-tumor T cell immunity in vivo.     

Toxicarioside A Inhibits Tumor Growth and Angiogenesis: Involvement of TGF-β/Endoglin Signaling

Toxicarioside A is a cardenolide isolated mainly from plants and animals. Emerging evidence demonstrate that cardenolides not only have cardiac effects but also anticancer effects. In this study, we used in vivo models to investigate the antitumor activities of toxicarioside A and the potential mechanisms behind them. Murine colorectal carcinoma (CT26) and Lewis lung carcinoma (LL/2) models were established in syngeneic BALB/c and C57BL/6 mice, respectively. We found that the optimum effective dose of toxicarioside A treatment significantly suppressed tumor growth and angiogenesis in CT and LL/2 tumor models in vivo. Northern and Western blot analysis showed significant inhibition of endoglin expression in toxicarioside A-treated human umbilical vein endothelial cells (HUVECs) in vitro and tumor tissues in vivo. Toxicarioside A treatment significantly inhibited cell proliferation, migration and invasion, but did not cause significant cell apoptosis and affected other membrane protein (such as CD31 and MHC I) expression. In addition, TGF-β expression was also significantly inhibited in CT26 and LL/2 tumor cells treated with toxicarioside A. Western blot analysis indicated that Smad1 and phosphorylated Smad1 but not Smad2/3 and phosphorylated Smad2/3 were attenuated in HUVECs treated with toxicarioside A. Smad1 and Smad2/3 signaling remained unchanged in CT26 and LL/2 tumor cells treated with toxicarioside A. Endoglin knockout by small interfering RNA against endoglin induced alternations in Smad1 and Smad2/3 signaling in HUVECs. Our results indicate that toxicarioside A suppresses tumor growth through inhibition of endoglin-related tumor angiogenesis, which involves in the endoglin/TGF-β signal pathway.     

Hsp110 over-expression increases the immunogenicity of the murine CT26 colon tumor

Several studies have suggested a positive correlation between heat shock protein (hsp) expression and tumor immunogenicity. Independently, many studies have shown that hsp purified from tumors can be used as a tumor-specific vaccine. In this study, we have explored the connection between hsp expression and anti-tumor immunity by transducing murine CT26 colon carcinoma cells with the cDNA of a major hsp, i.e. hsp110. We have shown that over-expression of hsp110 has no effect on CT26 tumor cell growth in vitro, and does not inhibit their anchorage-independent growth capacity. However, in situ, hsp110 over-expressing CT26 tumor (CT26-hsp110) grew at a significantly reduced rate as compared to the wild-type CT26 tumor in immunocompetent mice. Moreover, immunization of mice with inactivated CT26-hsp110 cells significantly inhibited the growth of wild-type CT26 tumor. This immunity was associated with an increased frequency of tumor-specific T cells after vaccination. An in vivo antibody depletion assay demonstrated that inactivated CT26-hsp110 cells elicited anti-tumor responses involving CD8(+) T cells and natural killer (NK) cells, but not CD4(+) T cells. Lastly, the effect of the addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to these vaccine formulations was determined. Mice immunized with irradiated CT26-hsp110 cells combined with GM-CSF-producing bystander cells revealed a complete inhibition of CT26 tumor growth, indicating a synergy between inactivated CT26-hsp110 vaccine activity and GM-CSF. These observations demonstrate that manipulation of hsp110 expression in tumors, specifically when combined with GM-CSF, represents a potentially powerful approach to cancer vaccine formulation.     

Anti-tumor activity of the combination of cetuximab, an anti-EGFR blocking monoclonal antibody and ZD6474, an inhibitor of VEGFR and EGFR tyrosine kinases

PURPOSE: The epidermal growth factor receptor (EGFR) autocrine pathway plays an important role in cancer cell growth. Vascular endothelial growth factor A (VEGF-A) is a key regulator of tumor-induced endothelial cell proliferation and vascular permeability. ZD6474 is an orally available, small molecule inhibitor of VEGF receptor-2 (VEGFR-2), EGFR and RET tyrosine kinase activity. We investigated the activity of ZD6474 in combination with cetuximab, an anti-EGFR blocking monoclonal antibody, to determine the anti-tumor activity of EGFR blockade through the combined use of two agents targeting the receptor at different molecular sites in cancer cells and of VEGFR-2 blockade in endothelial cells. EXPERIMENTAL DESIGN: The anti-tumor activity in vitro and in vivo of ZD6474 and/or cetuximab was tested in human cancer cell lines with a functional EGFR autocrine pathway. RESULTS: The combination of ZD6474 and cetuximab determined synergistic growth inhibition in all cancer cell lines tested as assessed by the Chou and Talalay method. In nude mice bearing established human colon carcinoma (GEO) or lung adenocarcinoma (A549) xenografts and treated with ZD6474 and/or cetuximab for 4 weeks, a reversible tumor growth inhibition was caused by each drug. In contrast, a more significant tumor growth delay resulted from the combination of the two agents with an approximately 100-110 days increase in mice median overall survival as compared to single agent treatment. CONCLUSIONS: This study provides a rationale for evaluating in a clinical setting the double blockade of EGFR in combination with inhibition of VEGFR-2 signaling as cancer therapy.     

Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma

Mitigation of regulatory T cell-mediated immunosuppression and elicitation of immunogenic tumor cell death are crucial events for optimal anti-tumor immune activity in vivo. This study was designed to investigate the potential synergistic activity of the combined use of cyclophosphamide (CP) and doxorubicin (DR), both of which are known to resolve these two issues. BALB/c mice were inoculated subcutaneously with CT-26 carcinoma cells in the bilateral flank and treated with an intraperitoneal injection of a low dose of CP followed by an intratumoral injection of DR into one side of the tumor. We found that, in addition to a significant suppression of growth on the DR-treated side of the tumor, combination therapy suppressed the growth of DR-untreated remote tumors in both tumor-specific and T cell-dependent manners. Mitomycin C showed no such synergistic anti-tumor activity with CP treatment. Combination therapy increased the frequency of interferon (IFN)-γ-producing T lymphocytes specific to a CT-26-associated class I-binding tumor peptide in the tumor-draining lymph nodes. Real-time PCR analysis revealed that combination therapy led to an increase in IFN-γ and tumor necrosis factor-α mRNA expression; however, levels of Foxp3 and transforming growth factor-β within the remote tumor tissues were decreased. In addition, knock down of calreticulin expression in CT-26 cells using small interfering RNA attenuated anti-tumor vaccine effects induced by DR-treated CT-26 cells. These results provide an immunological rationale for the combined use of chemotherapeutic drugs, i.e., CP and DR, and further recommend their use with current cancer vaccines.     

Biological and anti-vascular activity evaluation of ethoxy-erianin phosphate as a vascular disrupting agent

The effects of ethoxy-erianin phosphate (EBTP) on cell proliferation, mitotic cell arrest, migration, infiltration, and endothelial tubular structures were evaluated in this study. The antiproliferative activity of EBTP and combretastatin A-4P (CA4P) was analyzed on several tumor cells (including MCF-7, HeLa, 2LL, and 2LL-IDO) and on an endothelial cell (human umbilical vein endothelial cells [HUVECs]) as well as a human normal liver cell (L02). The results showed that EBTP possessed antiproliferative activity in the micromole range and was relatively less toxic than CA4P. Treating HUVECs with EBTP caused cell accumulation in the G2/M phase, and wound-healing assays indicated that EBTP inhibited cell migration. Furthermore, EBTP and CA4P destroyed the vasculature in endothelial cells and showed vascular disrupting activity of the chorioallantoic membrane in fertilized chicken eggs. In addition, we found that EBTP suppressed the expression of indoleamine 2,3-dioxygenase (IDO) and significantly inhibited IDO-induced migration and infiltration of 2LL-IDO cells. Administration of EBTP blocked vasculogenic mimicry in 2LL-IDO cells, which was typically observed in tube formation assays of 2LL-IDO cells. Moreover, the results of Lewis lung carcinoma in mice showed a high inhibition rate of EBTP. EBTP is an effective vascular disrupting agent that is superior to CA4P and may prevent and treat malignancy by inhibiting the expression of IDO.     

Early therapy evaluation of combined cetuximab and irinotecan in orthotopic pancreatic tumor xenografts by dynamic contrast-enhanced magnetic resonance imaging

Early pancreatic cancer response following cetuximab and/or irinotecan therapies was measured by serial dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) before and during therapy. Groups 1 to 4 (n = 6/group) of SCID mice bearing orthotopic pancreatic adenocarcinoma xenografts expressing luciferase were treated with phosphate-buffered saline, cetuximab, irinotecan, or cetuximab combined with irinotecan, respectively, twice weekly for 3 weeks. DCE-MRI was performed on days 0, 1, 2, and 3 after therapy initiation, whereas anatomic magnetic resonance imaging was performed on days 0, 1, 2, 3, 6, and 13. Bioluminescence imaging was performed on days 0 and 21. At day 21, all tumors were collected for further histologic analyses (Ki-67 and CD31 staining), whereas tumor dimensions were measured by calipers. The Ktrans values in the 0.5 mm-thick peripheral tumor region were calculated, and the changes in Ktrans during the 3 days posttherapy were compared to tumor volume changes, bioluminescent signal changes, and histologic findings. The Ktrans changes in the peripheral tumor region after 3 days of therapy were linearly correlated with 21-day decreases in tumor volume (p < .001), bioluminescent signal (p = .050), microvessel densities (p = .002), and proliferating cell densities (p = .001). This study supports the clinical use of DCE-MRI for pancreatic cancer patients for early assessment of an anti-epidermal growth factor receptor therapy combined with chemotherapy.     

Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity

Clostridium difficile toxin B (TcdB) is a key virulence factor of bacterium and induces intestinal inflammatory disease. Because of its potent cytotoxic and proinflammatory activities, we investigated the utility of TcdB in developing anti-tumor immunity. TcdB induced cell death in mouse colorectal cancer CT26 cells, and the intoxicated cells stimulated the activation of mouse bone marrow-derived dendritic cells and subsequent T cell activation in vitro. Immunization of BALB/c mice with toxin-treated CT26 cells elicited potent anti-tumor immunity that protected mice from a lethal challenge of the same tumor cells and rejected pre-injected tumors. The anti-tumor immunity generated was cell-mediated, long-term, and tumor-specific. Further experiments demonstrated that the intact cell bodies were important for the immunogenicity since lysing the toxin-treated tumor cells reduced their ability to induce antitumor immunity. Finally, we showed that TcdB is able to induce potent anti-tumor immunity in B16-F10 melanoma model. Taken together, these data demonstrate the utility of C. difficile toxin B for developing anti-tumor immunity.     

Photodynamic therapy of tumors can lead to development of systemic antigen-specific immune response

Background

The mechanism by which the immune system can effectively recognize and destroy tumors is dependent on recognition of tumor antigens. The molecular identity of a number of these antigens has recently been identified and several immunotherapies have explored them as targets. Photodynamic therapy (PDT) is an anti-cancer modality that uses a non-toxic photosensitizer and visible light to produce cytotoxic reactive oxygen species that destroy tumors. PDT has been shown to lead to local destruction of tumors as well as to induction of anti-tumor immune response.

Methodology/Principal Findings

We used a pair of equally lethal BALB/c colon adenocarcinomas, CT26 wild-type (CT26WT) and CT26.CL25 that expressed a tumor antigen, β-galactosidase (β-gal), and we treated them with vascular PDT. All mice bearing antigen-positive, but not antigen-negative tumors were cured and resistant to rechallenge. T lymphocytes isolated from cured mice were able to specifically lyse antigen positive cells and recognize the epitope derived from beta-galactosidase antigen. PDT was capable of destroying distant, untreated, established, antigen-expressing tumors in 70% of the mice. The remaining 30% escaped destruction due to loss of expression of tumor antigen. The PDT anti-tumor effects were completely abrogated in the absence of the adaptive immune response.

Conclusion

Understanding the role of antigen-expression in PDT immune response may allow application of PDT in metastatic as well as localized disease. To the best of our knowledge, this is the first time that PDT has been shown to lead to systemic, antigen- specific anti-tumor immunity.     

Use of photoacoustic imaging for monitoring vascular disrupting cancer treatments

Vascular disrupting agents disrupt tumor vessels, blocking the nutritional and oxygen supply tumors need to thrive. This is achieved by damaging the endothelium lining of blood vessels, resulting in red blood cells (RBCs) entering the tumor parenchyma. RBCs present in the extracellular matrix are exposed to external stressors resulting in biochemical and physiological changes. The detection of these changes can be used to monitor the efficacy of cancer treatments. Spectroscopic photoacoustic (PA) imaging is an ideal candidate for probing RBCs due to their high optical absorption relative to surrounding tissue. The goal of this work is to use PA imaging to monitor the efficacy of the vascular disrupting agent 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) through quantitative analysis. Then, 4T1 breast cancer cells were injected subcutaneously into the left hind leg of eight BALB/c mice. After 10 days, half of the mice were treated with 15 mg/kg of DMXAA and the other half were injected with saline. All mice were imaged using the VevoLAZR X PA system before treatment, 24 and 72 hours after treatment. The imaging was done at six wavelengths and linear spectral unmixing was applied to the PA images to quantify three forms of hemoglobin (oxy, deoxy and met-hemoglobin). After imaging, tumors were histologically processed and H&E and TUNEL staining were used to detect the tissue damage induced by the DMXAA treatment. The total hemoglobin concentration remained unchanged after treatment for the saline treated mice. For DMXAA treated mice, a 10% increase of deoxyhemoglobin concentration was detected 24 hours after treatment and a 22.6% decrease in total hemoglobin concentration was observed by 72 hours. A decrease in the PA spectral slope parameters was measured 24 hours after treatment. This suggests that DMXAA induces vascular damage, causing red blood cells to extravasate. Furthermore, H&E staining of the tumor showed areas of bleeding with erythrocyte deposition. These observations are further supported by the increase in TUNEL staining in DMXAA treated tumors, revealing increased cell death due to vascular disruption. This study demonstrates the capability of PA imaging to monitor tumor vessel disruption by the vascular disrupting agent DMXAA.     

采用活体成像技术监测肿瘤生长及转移模型的建立

目的采用活体成像技术监测稳定高表达荧光素酶报告基因的肿瘤细胞在小鼠体内生长及转移情况,为肿瘤治疗的药物研发提供新的有用工具。方法采用lipofectamine2000介导的基因转染方法,将pcDNA3．1 7Luc载体转染小鼠高转移乳腺癌细胞株4T1、EMT-6及结肠癌细胞株CT26,经G418抗性筛选及有限稀释法获得可稳定高表达荧光素酶的单克隆细胞;MTT法测定各转染细胞对不同化疗药物的抗性,并采用活体成像的方法检测各转染细胞在小鼠体内的成瘤和转移。结果获得了可稳定高表达荧光素酶基因的单克隆细胞株,该单克隆细胞株具有与亲本细胞系相同的对化疗药物的敏感性;将单克隆细胞株植入小鼠皮下,可采用活体成像技术准确监测肿瘤细胞体内生长及转移。结论采用活体成像技术构建的肿瘤动物模型是拓展肿瘤体内生长、转移及治疗相关研究的理想模型。     

Fasting protects against the side effects of irinotecan but preserves its anti-tumor effect in Apc15lox mutant mice

Irinotecan is a widely used topoisomerase-I-inhibitor with a very narrow therapeutic window because of its severe toxicity. In the current study we have examined the effects of fasting prior to irinotecan treatment on toxicity and anti-tumor activity. FabplCre;Apc^15lox/+ mice, which spontaneously develop intestinal tumors, of 27 weeks of age were randomized into 3-day fasted and ad libitum fed groups, followed by treatment with a flat-fixed high dose of irinotecan or vehicle. Side-effects were recorded until 11 days after the start of the experiment. Tumor size, and markers for cell-cycle activity, proliferation, angiogenesis, and senescence were measured. Fasted mice were protected against the side-effects of irinotecan treatment. Ad libitum fed mice developed visible signs of discomfort including weight loss, lower activity, ruffled coat, hunched-back posture, diarrhea, and leukopenia. Irinotecan reduced tumor size in fasted and ad libitum fed groups similarly compared to untreated controls (2.4 ± 0.67 mm and 2.4 ± 0.82 mm versus 3.0 ± 1.05 mm and 2.8 ± 1.08 mm respectively, P < 0.001). Immunohistochemical analysis showed reduced proliferation, a reduced number of vascular endothelial cells, and increased levels of senescence in tumors of both irinotecan treated groups. In conclusion, 3 days of fasting protects against the toxic side-effects of irinotecan in a clinically relevant mouse model of spontaneously developing colorectal cancer without affecting its anti-tumor activity. These results support fasting as a powerful way to improve treatment of colorectal carcinoma patients.     

Colon carcinoma cell growth is associated with prostaglandin E2/EP4 receptor-evoked ERK activation

Cyclooxygenase (COX) and its prostanoid metabolites have been implicated in the control of cell survival; however, their role as mitogens remains undefined. To better understand the role of prostanoids on cell growth, we used mouse colon adenocarcinoma (CT26) cells to investigate the role of prostaglandin E(2) (PGE(2)) in cell proliferation. CT26 cells express both COX1 and COX2 and metabolize arachidonic acid to PGE(2.) Treatment with indomethacin, or COX-selective inhibitors, prevents PGE(2) biosynthesis and CT26 cell proliferation. The anti-proliferative effects of COX inhibition are rescued specifically by treatment with PGE(2) or the EP4 receptor-selective agonist PGE(1)-OH via phosphatidylinositol 3-kinase/extracellular signal-regulated kinase (ERK) activation, thus providing a functional link between PGE(2)-induced cell proliferation and EP4-mediated ERK signaling. Indomethacin or COX2 inhibitors, but not COX1 inhibitors, reduced the size and number of CT26-derived tumors in vivo. These inhibitory effects are paralleled by marked declines in the levels of tumor PGE(2), suggesting that their anti-tumor effects are directly associated with the inhibition of COX2 enzymatic activity. The described anti-tumor effects of indomethacin are evident whether it is administered at the time of, or 7 days after, tumor cell injection, suggesting that it has tumor preventive and therapeutic actions. Furthermore, the observation that indomethacin increases the survival rates of tumor-bearing mice, even after withdrawal of the drug, indicates that its effects are long lasting and that it may be potentially useful for the prevention and the clinical management of human cancers.     

Superior anti-tumor protection and therapeutic efficacy of vaccination with allogeneic and semiallogeneic dendritic cell/tumor cell fusion hybrids for murine colon adenocarcinoma

Cancer immunotherapy by dendritic cell (DC)/tumor cell fusion hybrids (DC/TC hybrids) has been shown to elicit potent anti-tumor effects via the induction of immune responses against multiple tumor-associated antigens. In the present study, we compared the anti-tumor effects of vaccinating Balb/c mice (H-2^d) with CT26CL25 colon carcinoma cells that had been fused with either syngeneic DCs from Balb/c mice, allogeneic DCs from C57BL/6 mice (H-2^b) or semiallogeneic DCs from B6D2F1 mice (H-2^b/d). Preimmunization with either semiallogeneic or allogeneic DC/TC hybrids induced complete protection from tumor challenge, whereas mice preimmunized with syngeneic DC/TC hybrids were only partially protected (75% tumor rejection). The average number of pulmonary metastases after intravenous tumor injection decreased significantly following immunization with semiallogeneic or allogeneic DC/TC hybrids (8.3 ± 7.9 or 16.3 ± 3.5, mean ± SD) relative to syngeneic DC/TC hybrids (67.8 ± 6.3). These data demonstrate that vaccination with semiallogeneic DC/TC hybrids resulted in the greatest anti-tumor efficacy. Anti-tumor effects showed by in vivo studies were virtually accomplished by the frequency of induced CTLs specific to both gp70 and β-galactosidase assessed by using pentameric assay. Among the fusion vaccines tested, semiallogeneic DC/TC hybrids induced the highest ratio of Th1 cytokine IFN-γ to Th2 cytokine IL-10. In addition, allogeneic or semiallogeneic DC/TC hybrids elicited a significantly stronger NK activity than syngeneic DC/TC hybrids. These findings suggest that in clinical settings, DCs derived from a healthy donor (which are generally characterized as more semiallogeneic than allogeneic) may be more capable than autologous DCs of inducing promising anti-tumor effects in vaccinations with DC/TC hybrids.     

Vascular disrupting agent DMXAA enhances the antitumor effects generated by therapeutic HPV DNA vaccines

Antigen-specific immunotherapy using DNA vaccines has emerged as an attractive approach for the control of tumors. Another novel cancer therapy involves the employment of the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In the current study, we aimed to test the combination of DMXAA treatment with human papillomavirus type 16 (HPV-16) E7 DNA vaccination to enhance the antitumor effects and E7-specific CD8+ T cell immune responses in treated mice. We determined that treatment with DMXAA generates significant therapeutic effects against TC-1 tumors but does not enhance the antigen-specific immune responses in tumor bearing mice. We then found that combination of DMXAA treatment with E7 DNA vaccination generates potent antitumor effects and E7-specific CD8+ T cell immune responses in the splenocytes of tumor bearing mice. Furthermore, the DMXAA-mediated enhancement or suppression of E7-specific CD8+ T cell immune responses generated by CRT/E7 DNA vaccination was found to be dependent on the time of administration of DMXAA and was also applicable to other antigen-specific vaccines. In addition, we determined that inducible nitric oxide synthase (iNOS) plays a role in the immune suppression caused by DMXAA administration before DNA vaccination. Our study has significant implications for future clinical translation.     

Enhancement of hexokinase II inhibitor-induced apoptosis in hepatocellular carcinoma cells via augmenting ER stress and anti-angiogenesis by protein disulfide isomerase inhibition

3-bromopyruvate (3-BP), a hexokinase (HK) II inhibitor, promotes tumor cell death by inducing endoplasmic reticulum (ER) stress in human hepatocellular carcinoma (HCC) cell lines. Protein disulfide isomerase (PDI) is an essential folding catalyst and attenuates ER stress by folding the misfolded proteins. We examined if PDI is expressed in hypoxic HCC cells, and evaluated its inhibition potentiated HK II inhibitor-induced ER stress in hypoxic HCC cells. HCC apoptotic cell death was assessed by DAPI staining and apoptotic signaling pathways were explored by immunoblot analysis. An in vivo model of HCC was established in C3H mice intradermally with implanted MH134 cells. 3-BP with/without a PDI inhibitor (bacitracin) was subsequently administered. The anti-tumor efficacies were evaluated by measuring tumor volumes and quantifying apoptotic cells and microvessel densities (MVDs). HCC cells were found to express PDI in a hypoxia-inducible manner. The simultaneous treatment of bacitracin and 3-BP enhanced 3-BP-induced apoptosis. This enhancement was attributed to increased ER stress and JNK activation compared to the cells treated with just 3-BP. In an in vivo model of HCC, tumor growth was significantly suppressed in mice co-treated with bacitracin and 3-BP, and the percentages of apoptotic cells significantly increased and MVDs significantly decreased. These results demonstrated that PDI was induced in hypoxic HCC tissue and that PDI inhibition enhanced HK II inhibitor-induced anti-tumor efficacy synergistically via augmenting ER stress and anti-angiogenesis in vivo. Thus, blockage of PDI activity in combination with HK II inhibitor may be therapeutically useful in HCCs.     

Anticancer immunotherapy using inactivated Sendai virus particles

Ultraviolet-inactivated, replication-defective Sendai virus particles (Hemagglutinating virus of Japan envelope, HVJ-E) injected into murine colon carcinoma (CT26) tumors growing in syngeneic Balb/c mice eradicated 60-80% of the tumors and obviously inhibited the growth of the remainder. Induced adaptive anti-tumor immune responses were dominant in the tumor eradication process because the effect was abrogated in severe combined immunodeficient (SCID) mice. Murine and human dendritic cells (DCs) underwent dose-dependent maturation by HVJ-E in vitro. Profiles of cytokines secreted by DCs after HVJ-E stimulation showed that the amount of IL-6 released was comparable to that elicited by live HVJ. Real-time RT-PCR and immunohistochemistry revealed that HVJ-E induced a remarkable infiltration of DCs, CD4+ and CD8+ T cells into tumors and CT26 specific cytotoxic T lymphocytes (CTL) were induced. On the other hand, conditioned medium from DCs stimulated by HVJ-E (H-DCCM) rescued CD4+CD25- effector T cell proliferation from Foxp3+CD4+CD25+ regulatory T cell (Treg) mediated suppression and IL-6 was presumably dominant for this phenomenon. We also confirmed such rescue in mice treated with HVJ-E in vivo. Moreover, anti-tumor effect of HVJ-E was significantly reduced by an in vivo blockade of IL-6 signaling. Depending on cancer cell types, it is also expected that HVJ-E eradicates tumor by its direct cytotoxity against cancer cells or activating NK cells. Because it can enhance anti-tumor immunity and simultaneously remove Treg mediated suppression, HVJ-E shows promise as a novel therapeutic for cancer immunotherapy.