S180 Sarcoma Research Articles

Selective self-induced stimulus amplification prodrug platform for inhibiting multidrug resistance and lung metastasis

Tumor heterogeneity is considered as one of main obstacles to limit the clinical application of stimuli-responsive nanocarriers. Multidrug resistance (MDR) is also a major challenge in cancer chemotherapy. Here, we developed a tumor redox heterogeneity-responsive prodrug with self-induced reactive oxygen species (ROS) amplification property for facilitating rapid drug release and overcoming MDR and lung metastasis. The prodrug can self-assemble into polymer micelles (PMs) with high drug loading content (~30%), good physiological stability, prolonged systemic circulation and enhanced tumor distribution. Moreover, the prodrug PMs can stimulate tumor-specific ROS signal amplification, which provided a replenishment of consumed ROS necessary for rapid and complete drug release. The elevated ROS could not only evoke the mitochondria-dependent apoptosis by caspase-9/3 activation, but also inhibit inherent and acquired drug resistance by altering expression of Bcl-2 protein family and by reducing mitochondria membrane potential (ΔΨm) and ATP level in cancer cells. As a result, the prodrug PMs showed enhanced efficacy for inhibiting tumor growth in S180 sarcoma tumor model and in drug-resistant tumor model MCF-7/ADR and preventing lung metastasis in 4T1 in situ breast cancer model. This novel approach reported here may provide a promising strategy in the design of stimuli-responsive nanocarriers for efficient therapy of multidrug resistant and metastatic tumor.

Dielectrophoresis Microfluidic Enrichment Platform with Built-In Capacitive Sensor for Rare Tumor Cell Detection

The manipulation and detection of rare cells are important for many applications in early disease diagnosis and medicine. This study presents a dielectrophoresis (DEP) microfluidic enrichment platform combined with a built-in capacitive sensor for circulating tumor cell detection. The microchip is composed of a lollipop-shaped gold microelectrode structure under a polydimethylsiloxane chamber. A prototype of the device was fabricated using standard micromachining technology. With the proposed device, target cells (in this study, A549 non-small human lung carcinoma and S-180 sarcoma cell lines) are firstly guided toward the center of the working chamber via DEP forces. Then, the target cells are captured by an electrode immobilized by anti-EGFR, which has high affinity toward the target cells. After the cell concentration process, the differential capacitance is read to detect the presence of the target cells. Numerical simulations and measurement experiments were performed to demonstrate the high sensitivity of differential capacitive sensing. The obtained results show high sensitivity for S-180 cell detection (3 mV/cell). The proposed platform is suitable for rapid cancer diagnoses and other metabolic disease applications.

Anti-tumour effects of polysaccharide extracted from Acanthopanax senticosus and cell-mediated immunity.

Acanthopanax senticosus, also known as Siberian ginseng, is widely distributed throughout northern Asia and used in traditional Chinese medicine; it has been reported to prevent a number of diseases. However, the association between the antitumour and immunostimulatory activities of polysaccharide extracted from A. senticosus (ASPS) remains to be elucidated. The aim of the present study was to investigate the anti-tumour and immunomodulatory effects of polysaccharide extracted from ASPS on Crocker sarcoma S180, hepatic carcinoma H22 and uterine cervical carcinoma U14 tumour cell lines implanted in mice. High performance liquid chromatography, gas chromatography and infrared spectroscopy were used to analyse the monosaccharide composition of ASPS. The monosaccharide composition of ASPS (Arabic candy: Xylose: Glucose: Mannose) was 7.1:22.3:7.6:1.0. On day 0, female Kunming mice, were injected subcutaneously with 1×108 tumour cells in 0.2 ml. The inoculated mice were subsequently divided into five groups (10 mice/group) as follows: Model group, treated with normal saline; positive control group, treated with 30 mg/kg cyclophosphamide (CTX); and three treatment groups, treated with 200, 100 or 50 mg/kg ASPS. Non-inoculated mice were divided into the normal group, which was treated with normal saline, and the negative control group, which was treated with 200 mg/kg ASPS (n=10/group). CTX and ASPS were administered intragastrically once daily for 10 days. All mice were sacrificed on day 11. ASPS was observed to have an inhibitory effect on the growth of S180, H22 and U14 cells in solid and ascites tumour-bearing mice. Serum interleukin (IL)-2 and IL-12 levels were significantly increased in S180 solid tumour-bearing mice treated with 200 or 100 mg/kg ASPS compared with mice in the normal, control and model groups (P<0.05), whereas serum IL-2 and IL-12 levels were significantly decreased in the cyclophosphamide treatment group compared with the normal, control and model groups (P<0.05). No significant difference in serum levels of tumour necrosis factor-α level was observed between any groups. In S180 and U14 solid tumour-bearing mice, no significant differences in serum levels of interferon (INF)-γ level in were observed between groups; however, in H22 solid tumour-bearing mice, treatment with ASPS significantly increased serum INF-γ compared with the positive control group (P<0.05). The results may provide a basis for the potential application of ASPS in clinical treatment for cancer.

Structural Features, Antitumor and Antioxidant Activities of Rice Bran Polysaccharides Using Different Extraction Methods.

Polysaccharides is extracted from rice bran (RBP) using hydrothermal, microwave-assisted and enzyme-assisted extraction methods. The results suggested that the antitumor activity of RBP was associated with enhancement of immunization and antioxidant. RBP could be explored as a natural antitumor and antioxidant agent applied in medicines and functional foods.

Antitumor activity of yulangsan polysacchrides in mice bearing S180 sarcoma tumors.

Sarcoma is one of the most prevalent pediatric tumors and the therapeutic role of chemotherapy has yet to be elucidated. It has been reported that extracts of Longyanshen (Yulangsan) may enhance the sensitivity of drug-resistant cancer cell lines, and improve the immune dysfunction induced by cyclophosphamide (CTX) in mice. The present in vivo study investigated the antitumor effects of Yulangsan polysaccharides (YLSPS) and their interaction with CTX in murine sarcoma 180 (S180)-bearing mice. Immunohistochemistry was used to detect the expression of apoptosis-related proteins. The ultrastructure of sarcoma cells was examined by transmission electron microscopy and the tumor growth rate was determined by measuring the tumor weight. A dose-dependent inhibition of sarcoma growth was observed in S180-bearing mice following administration of YLSPS. In combination with CTX, an additive antitumor effect was obtained, which was accompanied by amelioration of immune function. YLSPS also potentiated the tumor suppression effect of CTX while avoiding cytotoxicity against immune cells. YLSPS inhibited sarcoma growth in S180-bearing mice through the induction of apoptosis in S180 sarcoma cells. YLSPS also attenuated CTX-induced cytotoxicity to the immune system while potentiating the tumor suppression effect. These results provide additional information regarding combination therapy with YLSPS and chemotherapy for the treatment of sarcoma.

Abstract 166: Recombinant protein MBP-NAP restricts tumor progression by triggering T-cell immunity in mouse metastatic melanoma model

Abstract The pro-inflammatory and immunomodulatory properties of Helicobacter pylori neutrophil activating protein (Hp-NAP) not only make it play an important role in disease pathogenesis, but also make it a potential candidate for applications, including vaccine and drug development. Our previous work demonstrated that the recombinant Hp-NAP fused with the maltose-binding protein of Escherichia coli (rMBP-NAP) exert an important role in regulating the differentiation of Th1 cells. As a potential TLR2 ligand, it was reported to possess the ability to induce systemic antitumor immunity in murine hepatoma H22 and sarcoma S180 tumor models. To further understand the antitumor and immunomodulatory effect of rMBP-NAP, we elucidate the effect and mechanism of rMBP-NAP at the local immune response modulation in established mouse B16-F10 melanoma pulmonary metastasis model. Our results demonstrated that metastatic lung tumor growth was significantly arrested after rMBP-NAP treatment, along with marked reduction in the number of lung nodules and significant increase in survival. Flow cytometry immunophenotyping and Quantitative RT-PCR analyses demonstrated that rMBP-NAP could induce both local and systemic immune responses, which associated with higher influx of CD3+CD4+T cells, CD3+CD8+T cells and higher secretion of interferon (IFN)-γ and interleukin (IL)-27 cytokines. The intraperitoneal administration of rMBP-NAP in mice promoted infiltration of lymphocyte in the lungs and reduced the production of several proinflammatory cytokines, such as IL-6 and transforming growth factor beta (TGF) -β, indicating an anti-inflammatory effect in local area. By comparing with control mice, mRNA expression of chemokines also revealed that rMBP-NAP treatment substantially decreased the expression of CCL2 and CCL20 in tumor tissues, which are central to recruit myeloid-derived suppressor cell (MDSC) and regulatory T cells (Treg) to tumor microenviroment and form immunosuppression. Moreover, we assess the expression of vascular marker CD34 in pulmonary section using immunohistochemical method, the results showed the vascularization was apparently inhibited in rMBP-NAP treated tumor bearing mouse. In conclusion, rMBP-NAP could induce the activation of effective Th1/Tc1 cells and the production of relevant inflammatory cytokines and chemokines, which are responsible for enhencing T cell immunity and reversing immunosuppression against metastatic cancer progression. Our findings strongly indicate that rMBP-NAP treatment might be a novel therapeutic approach against metastatic melanoma, and rMBP-NAP might be a potential template for the development of agents that could be used as immunomodulatory adjuvant in the therapy against melanoma. Note: This abstract was not presented at the meeting. Citation Format: Ting Wang, Cong Ding, Zhenyu Ji, Xin Liu, Mingxuan Du, Qiaozhen Kang. Recombinant protein MBP-NAP restricts tumor progression by triggering T-cell immunity in mouse metastatic melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 166. doi:10.1158/1538-7445.AM2017-166

Novel, nano-sized, liposome-encapsulated polyamidoamine dendrimer derivatives facilitate tumour targeting by overcoming the polyethylene glycol dilemma and integrin saturation obstacle

Drug delivery systems (DDSs) commonly employ arginine–glycine–aspartic acid (RGD) peptides with polyethylene glycol (PEG)-dependent enhanced permeability and retention (EPR) effect to optimise tumour-targeting. However, the PEG dilemma and integrin saturation obstacle are major challenges. To address these issues, we constructed a novel, nano-sized DDS by encapsulating doxorubicin (DOX)-loaded folic acid derivatives of polyamidoamine dendrimer (PAMAM G5.0) in cyclic RGD-tyrosine-lysine pentapeptide (c[RGDyK])-modified liposomes (RGD-SL[FND/DOX]), prepared using thin-film hydration, film-dispersion and hydration-sonication. The liposomes were PEGylated, sterically stabilised and pH-sensitive. In vitro, RGD-SL[FND/DOX] showed pH-sensitive holistic FND/DOX release, and pH-dependent uptake and cytotoxicity in human cancer KB cells. At pH 7.4, RGD-SL[FND/DOX] demonstrated greater cellular uptake and cytotoxicity than relevant control formulations (except FND/DOX) did, although this advantage disappeared at pH 6.5. In vivo, RGD-SL[FND/DOX] inhibited S180 sarcoma xenografted tumour growth in Kunming mice more effectively than FND/DOX did. These findings demonstrate the feasibility of constructing double-stage tumour-targeting nano-sized DDSs such as RGD-SL[FND/DOX]. c[RGDyK] and the EPR effect, facilitated by the particle size (about 110 nm) and PEGylation, helped to target the DDS to the tumour tissue, while the subsequent pH-dependent release of FND/DOX and folic acid-mediated endocytosis specifically targeted the tumour cells, thereby overcoming the PEG dilemma and integrin saturation obstacle.

Granulocyte-macrophage colony-stimulating factor increases tumor growth and angiogenesis directly by promoting endothelial cell function and indirectly by enhancing the mobilization and recruitment of proangiogenic granulocytes.

Granulocyte-macrophage colony-stimulating factor has been widely used as an adjuvant therapy for cancer patients exhibiting myelosuppression induced by chemotherapy or radiotherapy. However, the effects of granulocyte-macrophage colony-stimulating factor on tumor growth, as well as its precise mechanism, are still controversial due to inconsistent evidence. This study investigated the effect of exogenous granulocyte-macrophage colony-stimulating factor on the growth of B16 melanoma, S180 sarcoma, and U14 cervical carcinoma in mice. The angiogenesis and recruitment of bone-marrow-derived cells were analyzed in tumor tissues. Interactions among granulocyte-macrophage colony-stimulating factor, bone-marrow-derived cells, and B16 tumor cells were investigated in vitro. Proangiogenic types of bone-marrow-derived cells in blood were assessed both in vivo and in vitro. The results showed that granulocyte-macrophage colony-stimulating factor markedly facilitated the growth of B16 and S180 tumors, but not U14 tumors. Granulocyte-macrophage colony-stimulating factor increased the densities of blood vessels and the number of bone-marrow-derived cells in B16 tumor tissues. The granulocyte-macrophage colony-stimulating factor-induced enhancement of tumor cell proliferation was mediated by bone-marrow-derived cells in vitro. Meanwhile, a distinct synergistic effect on endothelial cell function between granulocyte-macrophage colony-stimulating factor and bone-marrow-derived cells was observed. After separating two types of bone-marrow-derived cells, granulocyte-macrophage colony-stimulating factor-induced enhancement of tumor growth and angiogenesis in vivo was mediated by proangiogenic cells in granulocytes, but not monocytes, with CD11b+, vascular endothelial growth factor receptor 2, and C-X-C chemokine receptor 4 granulocytes possibly involved. These data suggest that granulocyte-macrophage colony-stimulating factor contributes to the growth and angiogenesis of certain types of tumor, and these mechanisms are probably mediated by proangiogenic cells in granulocytes. Applying granulocyte-macrophage colony-stimulating factor may attenuate the antitumor effects of chemotherapy and radiotherapy in certain types of tumor.

Synergistic antitumor efficacy of antibacterial helvolic acid from Cordyceps taii and cyclophosphamide in a tumor mouse model.

The antibacterial agent helvolic acid, which was isolated from the active antitumor fraction of Cordyceps taii, showed potent cytotoxicity against different human cancer cells. In the present study, the invivo antitumor effect of helvolic acid was investigated in murine sarcoma S180 tumor-bearing mice. Doses of 10 and 20 mg/kg/day helvolic acid did not exert significant antitumor activity. Interestingly, co-administration of 10 mg/kg/day helvolic acid and 20 mg/kg/day cyclophosphamide (CTX) - a well-known chemotherapy drug - showed promising antitumor activity with a growth inhibitory rate of 70.90%, which was much higher than that of CTX alone (19.5%). Furthermore, the combination markedly prolonged the survival of tumor-bearing mice. In addition, helvolic acid enhanced the immune organ index. The protein expression levels of β-catenin, cyclin D1, and proliferating cell nuclear antigen were significantly suppressed in mice treated with 20 mg/kg/day helvolic acid and in those receiving combination therapy. Taken together, these results indicated that helvolic acid in combination with CTX showed potent invivo synergistic antitumor efficacy, and its mechanism of action may involve the Wnt/ β-catenin signaling pathway.

Aspirin may inhibit angiogenesis and induce autophagy by inhibiting mTOR signaling pathway in murine hepatocarcinoma and sarcoma models.

Aspirin is known to have inhibitory effects on growth development in various types of tumor. In previous studies, it was observed to inhibit angiogenesis by downregulating the expression of vascular endothelial growth factor-A (VEGF-A). In the present study, murine H22 hepatocarcinoma and S180 sarcoma models were used to ascertain whether aspirin could inhibit angiogenesis and promote autophagy in tumors. Tumor-bearing mice were randomly divided into four groups with 10 mice per group: i) no treatment; ii) low-dose aspirin (100 mg/kg); iii) high-dose aspirin (400 mg/kg); iv) everolimus group (4 mg/kg). The effects of high-dose aspirin were validated through preliminary experiments. The drug treatment was administered every day for 14 days. The tumor size was measured every other day and then the tumor growth curve was plotted, and the tumor inhibitory rates were calculated. The expression levels of phosphorylated mammalian target of rapamycin (p-mTOR), hypoxia-inducible factor-1α (HIF-1α), VEGF-A, UNC-51-like kinase-1 (ULK1) and microtubule-associated protein 1 light chain 3A (LC3A) were detected by immunohistochemistry and western blot analysis, respectively. We observed that tumor growth delay was achieved in both H22 hepatocarcinoma and S180 sarcoma models following treatment with aspirin. The tumor growth inhibition rates induced by low and high-dose aspirin and everolimus were 19.6, 33.6 and 53.7% (P<0.05) in H22 hepatocarcinoma, and 25.7, 40.6 and 48.7% (P<0.05) in S180 sarcoma. The immunohistochemistry and western blot analysis data from the models revealed that the expression of p-mTOR, HIF-1α and VEGF-A was decreased, while the expression of ULK1 and LC3A was increased following treatment with aspirin and everolimus. The changes were more apparent in the high-dose aspirin and everolimus groups (P<0.01). The inhibitory action of aspirin and everolimus on tumor angiogenesis may be through inhibiting the expression of p-mTOR, HIF-1α and VEGF-A. Alternatively, aspirin may induce autophagy by inhibiting the mTOR signaling target and then increasing ULK1 and LC3A.

The in Vitro and in Vivo Antitumor Activities of Tetracyclic Triterpenoids Compounds Actein and 26-Deoxyactein Isolated from Rhizome of Cimicifuga foetida L.

Aims: This work aims to study the in vitro and in vivo antitumor activities of tetracyclic triterpenoids compounds actein and 26-deoxyactein. Further, the mechanism is investigated. Methods: In vitro, a modified MTT method was used to assay the cytotoxicities of actein and 26-deoxyactein in 12 human tumor cell lines. In vivo, mouse sarcoma S180 and human lung cancer A549 cells were respectively implanted subcutaneously in ICR mice and nude mice to establish implanted tumor models. Flow cytometry (FCM) was used to assay the cycle distribution of the tumor cells. Immunohistochemistry was used to measure CD31-positive expression in the xenogrft tumor by analyzing microvessel density (MVD). In addition, acute toxicities of actein and 26-deoxyactein were also evaluated. Results: Actein and 26-deoxyactein inhibited the proliferation of the 12 human cancer cell lines tested with the values of 50% inhibitory concentrations (IC50) between 12.29 and 88.39 μg/mL. In vivo, both actein (3–27 mg/kg) and 26-deoxyactein (3–27 mg/kg) significantly inhibited the growth of the implanted sarcoma S180 in a dose-dependent manner. Actein (10, 30 mg/kg) and 26-deoxyactein (10, 30 mg/kg) markedly inhibited the xenograft growth with T/C (%) values of 38%, 55% for actein, and 35%, 49% for 26-deoxyactein. Compared with the vehicle control, actein (10, 30 mg/kg) and 26-deoxyactein (10, 30 mg/kg) significantly reduced the MVD in the xenograft tumor. The FCM result showed that human leukemia HL-60 cells were arrested at G1 phase after treated with either actein (6.25–25 μg/mL) or 26-deoxyactein (6.25–25 μg/mL) for 48 h. A limited trial in mice showed that both of the minimal lethal doses (MLDs) of actein and 26-deoxyactein were over 5 g/kg. Conclusions: Both actein and 26-deoxyactein have low toxicities. Importantly, both these two tetracyclic triterpenoids compounds isolated from rhizome of Cimicifuga foetida L. have significant antitumor activities in vitro and in vivo, which is associated with cell cycle arrest and angiogenesis inhibition.

Construction of random tumor transcriptome expression library for creating and selecting novel tumor antigens.

Novel tumor antigens are necessary for the development of efficient tumor vaccines for overcoming the immunotolerance and immunosuppression induced by tumors. Here, we developed a novel strategy to create tumor antigens by construction of random tumor transcriptome expression library (RTTEL). The complementary DNA (cDNA) from S180 sarcoma was used as template for arbitrarily amplifying gene fragments with random primers by PCR, then ligated to the C-terminal of HSP65 in a plasmid pET28a-HSP for constructing RTTEL in Escherichia coli. A novel antigen of A5 was selected from RTTEL with the strongest immunotherapeutic effects on S180 sarcoma. Adoptive immunotherapy with anti-A5 sera also inhibited tumor growth, further confirming the key antitumor roles of A5-specific antibodies in mice. A5 contains a sequence similar to protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1). The antisera of A5 were verified to cross-react with PCMT1 by Western blotting assay and vice versa. Both anti-A5 sera and anti-PCMT1 sera could induce antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity toward S180 cells by in vitro assay. Further assay with fluorescent staining showed that PCMT1 is detectable on the surface of S180 cells. Summary, the strategy to construct RTTEL is potential for creating and screening novel tumor antigens to develop efficient tumor vaccines. By RTTEL, we successfully created a protein antigen of A5 with significant immunotherapeutic effects on S180 sarcoma by induction of antibodies targeting for PCMT1.

Modulation of MMP-2 and TIMP-2 by low dose radiation in mice bearing S180 sarcoma

Abstract Objective To investigate the inhibition of low dose radiation (LDR) on S180 sarcomas and its modulation of MMP-2 and TIMP-2 in mice. Methods S180 subcutaneously implanted tumor model mice were randomly divided into two groups: control (N) and low dose radiation (LDR) groups. N mice were sacrificed after 12 h, whereas LDR mice were sacrificed after 12 (LDR-12 h), 24 (LDR-24 h), 48 (LDR-48 h), and 72 (LDR-72 h) h. Thereafter, we measured the tumor volumes. Histopathology was performed, and P-V immunohistochemistry was applied to assess MMP-2 and TIMP-2 expression. Results Compared with the control group, the tumor growth was significantly inhibited in the LDR groups (P &lt; 0.05). MMP-2 expression was considerably reduced in LDR-24h (P &lt; 0.05) and LDR-48h (P &lt; 0.05), whereas the change of TIMP-2 was not obvious in the LDR groups (P &gt; 0.05) in contrast to that of the control group. Conclusion LDR can effectively suppress the growth of S180 implanted tumors by reducing MMP-2, which is associated with invasion and metastasis.

Synthesis, radiolabeling and biological evaluation of butene amine oxime containing nitrotriazole as a tumor hypoxia marker

99mTc-BnAO, as a nonnitroaromatic hypoxia marker, is the subject of intensive research in recent years. In this study, a butene amine oxime–nitrotriazole (BnAO–NT) was synthesized and radiolabeled with 99mTc in high yield. Cellular uptakes of 99mTc-BnAO–NT and 99mTc-BnAO were tested using murine sarcoma S180 and hepatoma H22 cell lines. The highest hypoxic cellular uptake of 99mTc-BnAO–NT was 27.11 ± 0.73 and 14.85 ± 0.83 % for the S180 and H22 cell lines, respectively, whereas the normoxic cellular uptake of the complex was about 4–8 % for both cell lines. For 99mTc-BnAO, the highest hypoxic cellular uptake was 30.79 ± 0.44 and 9.66 ± 1.20 % for the S180 and H22 cell lines, respectively, while the normoxic cellular uptake was about 5 % for both cell lines. Both 99mTc-BnAO–NT and 99mTc-BnAO complexes showed hypoxic/normoxic differentials in the two cell lines, but the results were more significant for the S180 cell line. The in vitro results suggested that S180 may be better than H22 cell line in hypoxic biological evaluation of BnAO complexes. The biodistribution study was tested using a S180 tumor model. The complex 99mTc-BnAO–NT showed a selective enrichment in tumor tissues: At 4 h, the tumor-to-muscle ratio was 3.79 ± 0.98 and the tumor-to-blood ratio was 2.31 ± 0.34. Compared with the results of 99mTc-BnAO, the latter was at the same level. In vitro and in vivo studies demonstrated that 99mTc-BnAO–NT could be a hypoxia-sensitive radiotracer for monitoring hypoxic regions in a sarcoma S180 tumor.

Gene expression response to ascorbic acid in mice implanted with sarcoma S180 cells

Gene expression response to ascorbic acid in mice implanted with sarcoma S180 cells

Anti-tumor and immunomodulatory activity of iron hepta-tungsten phosphate oxygen clusters complex

Polyoxometalates (POMs) have attracted a considerable attention due to their unique structural characteristics, physicochemical properties and biological activities. In this study, iron hepta-tungsten phosphate oxygen clusters complex Na12H[Fe(HPW7O28)2]·44H2O (IHTPO) was synthesized and evaluated for in vitro cytotoxic activities on human hepatoma HepG2, leukemia K562, lung carcinoma A549, and large cell lung cancer NCI-H460 cells, therapeutic efficacies on mice transplantable tumor, and immunomodulatory potentials on the immune response in tumor-bearing mice. IHTPO exhibited lower in vitro cytotoxic activities against four human tumor cell lines, with the IC50 values being higher than 62.5μM (ca. 300μg/ml). IHTPO, however, significantly inhibited the growth of S180 sarcoma transplanted in mice. It was further showed that IHTPO could not only significantly promote splenocytes proliferation, NK cell and CTL activity from splenocytes, but remarkably enhance serum antigen-specific IgG, IgG2a and IgG2b antibody levels in S180-bearing mice. IHTPO also significantly promoted Th1 cytokines IFN-γ and IL-2 production, and up-regulated the mRNA expression levels of IFN-γ, IL-2 and Th1 transcription factors T-bet and STAT-4 in splenocytes from the S180-bearing mice. These results suggested that IHTPO significantly inhibited the growth of mice transplantable tumor, and that its in vivo antitumor activity might be achieved by improving Th1 protective cell-mediated immunity. IHTPO could act as antitumor agent with immunomodulatory activity.

Kit formulation for preparation and biological evaluation of a novel 99mTc-oxo complex with metronidazole xanthate for imaging tumor hypoxia

IntroductionAchieving an ideal 99mTc labeled nitroimidazole hypoxia marker is still considered to be of great interest. Metronidazole xanthate (MNXT) ligand was synthesized and radiolabeled with 99mTc-glucoheptonate (GH) to form the 99mTcO-MNXT complex, for the potential use as a novel probe for imaging tumor hypoxia. MethodsFor labeling, 99mTcO-MNXT was prepared by ligand-exchange reaction with 99mTc-GH. The radiochemical purity of the 99mTcO-MNXT complex was measured by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The distribution coefficient and stability of the complex was investigated. The structure of the 99mTcO-MNXT complex was verified by preparation and characterization of the corresponding stable rhenium complex. The cellular uptake of the 99mTcO-MNXT complex was determined in murine sarcoma S180 cell lines under hypoxic and aerobic conditions. The biodistribution and single photon emission computed tomography (SPECT) image studies of the 99mTcO-MNXT complex were performed in mice bearing S 180 tumor. ResultsThe radiochemical purity of the 99mTcO-MNXT complex was over 90%. It had good in vitro stability and its distribution coefficient indicated that it was a hydrophilic complex. When 99mTc and Re complexes were coinjected in HPLC, both radioactivity (for 99mTc complex) and UV detectors (for Re complex) showed nearly identical HPLC profiles, suggesting their structures are similar. The tumor cell experiment and the biodistribution in mice bearing S 180 tumor showed that the 99mTcO-MNXT complex had a good hypoxic selectivity and accumulated in the tumor with high uptake and good retention. Single photon emission computed tomography (SPECT) image studies showed that the tumor detection was observable. Conclusions99mTcO-MNXT is prepared from a kit without the need for purification and shows high tumor uptake, tumor/blood and tumor/muscle ratios, suggesting that it would be a promising candidate for imaging tumor hypoxia.

Antitumor and immunomodulatory effects of recombinant fusion protein rMBP-NAP through TLR-2 dependent mechanism in tumor bearing mice

The pro-inflammatory and immunomodulatory properties of Helicobacter pylori neutrophil activating protein (Hp-NAP) not only make it to play an important role in disease pathogenesis but also make it to be a potential candidate for therapeutic applications, including vaccine and drug development. Our previous work demonstrated that the recombinant Hp-NAP fused with the maltose binding protein of Escherichia coli (rMBP-NAP) play an important role in regulating the differentiation of Th1 cells. In this study, we investigated the ability of rMBP-NAP to induce antitumor immunity using two murine models of hepatoma H22 and sarcoma S180. Subcutaneous administration of mice with rMBP-NAP resulted in an about 40%–50% decrease of tumor growth compared with that of the control mice. Splenocytes from the tumor-bearing mice treated with rMBP-NAP showed a significant accumulation of CD4+ IFN-γ-secreting cells, which is a cytokine profile of Th1 cells. Furthermore, intravenous injection of T2.5, toll like receptor (TLR) 2 blocking antibody, significantly recede the antitumor effect of rMBP-NAP and the production of IFN-γ induced by rMBP-NAP. Our findings indicate that potentiality of rMBP-NAP to be a candidate for the development of immunomodulatory antitumoral drugs.

Anti-tumor effect investigation of obtustatin and crude Macrovipera lebetina obtusa venom in S-180 sarcoma bearing mice

Over the last few decades, research on snake venom toxins has provided not only new tools to decipher molecular details of various physiological processes, but also inspiration to design and develop a number of therapeutic agents. Isolated from the venom of Macrovipera lebetina obutusa (MLO), obtustatin represents the shortest known snake venom monomeric disintegrin specific inhibitor of α1β1 integrin. This low molecular weight peptide revealed a potent therapeutic effect on melanoma progression. Its oncostatic effect was related to the inhibition of angiogenesis. The aim of the proposed investigation was to study the influence of obtustatin and crude MLO venom on the S-180 sarcoma growth in vitro and in vivo. A S-180 sarcoma bearing mouse model, histological examination, DNA retardation assay were utilized to investigate the anti-tumor effects of MLO and obtustatin. In addition, some biochemical tests (chemiluminescence-ChL, TBA-test) were applied to elucidate the influence of obtustatin and crude MLO venom on the S-180 sarcoma. The size of tumor was significantly inhibited by MLO venom and obtustatin with the inhibitory rate of 50% and 33% at the doses of 10µg/mouse and 1mg/kg/day respectively. Both ChL and MDA decrease in the two treated groups. Both obtustatin and MLO venom have an anticancer activity and might be candidates for the treatment of malignant sarcoma. All our results have shown that both obtustatin and MLO venom have an anticancer activity and might be candidates for the treatment of malignant sarcoma.

Screening antitumor bioactive fraction from Sauromatum giganteum (Engl.) Cusimano & Hett and sensitive cell lines with the serum pharmacology method and identification by UPLC-TOF-MS.

Sauromatum giganteum (Engl.) Cusimano & Hett Tuber are used in Chinese folklore medicine for treatment of neoplasms. However, the claim has not been scientifically validated. The aim of the study is to screen the antitumor bioactive fraction of Sauromatum giganteum (Engl.) Cusimano & Hett Tuber and sensitive tumor cell lines using a cytotoxicity assay in vitro and tumor transplantation method in vivo, to support its use in folk medicine. The petroleum ether fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction were successively extracted by turn by the maceration under reflux assay. Screening of antitumor bioactive fraction and sensitive cell lines were measured by MTT assay and the serum pharmacology method, and in vivo the antitumor activities of the active fraction was evaluated by using S180 or H22 tumor-bearing mice model and Kunming mice. The active constituents of ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett were characterized by UPLC-TOF-MS. Compared with control groups, mice serum containing ethyl acetate fraction had a inhibition effect on SMMC-7721 cell, SGC-7901 cell, MCF-7 cell, HeLa cell, A549 cell, HT-29, and MDA-MB-231, respectively, but mice serum containing other four fractions had no different with that of control group. The inhibition capabilities of mice serum containing ethyl acetate fraction on the seven cell lines in descending order is SGC-7901 > SMMC-7721 > MCF-7 > HT-29 > A549 > HeLa > MDA-MB-231. In vivo the inhibition rate of 106, 318, 954 mg/kg·d ethyl acetate fraction dry extract to sarcoma S180 is 15.22%, 26.15% and 40.24%, respectively, and life prolonging rate to hepatoma H22 is 33.61%, 40.16% and 55.74%. A total of 14 compounds were identified in the ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett. The results of the experimental studies proved the antitumor activity of Sauromatum giganteum (Engl.) Cusimano & Hett and supported the traditional use of this plant. These data indicate the potential for the use of ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett Tuber in tumor therapy, anti-tumor activity on cancer cell line in descending order is SGC-7901 > SMMC-7721 > MCF-7 > HT-29 > A549 > HeLa > MDA-MB-231.