3LL Cell Research Articles

Co-encapsulation of fisetin and cisplatin into liposomes: Stability considerations and in vivo efficacy on lung cancer animal model

Lung cancer is a highly vascularized tumor for which a combination between an antitumor agent, cisplatin, and an antiangiogenic molecule, fisetin, appears a promising therapeutic approach. In order to deliver both chemotherapies within the tumor, to enhance fisetin solubility and decrease cisplatin toxicity, an encapsulation of both drugs into liposomes was developed. Purification and freeze-drying protocols were optimized to improve both the encapsulation and liposome storage. The cytotoxicity of the encapsulated chemotherapies was evaluated on Lewis lung carcinoma (3LL) cell lines. The antitumor effect of the combination was evaluated in vivo on an ectopic mouse model of Lewis Lung carcinoma. The results showed that fisetin and cisplatin co-loaded liposomes were successfully prepared. Freeze-drying allowed a 30 days storage limiting the release of both drugs. The combination index between liposomal fisetin and liposomal cisplatin on 3LL cell line after 24 h of exposure showed a clear synergism: CI = 0.7 for the co loaded liposomes and CI = 0.9 for the mixture of cisplatin loaded and fisetin loaded liposomes. The co-encapsulating formulation showed in vivo efficacy against an ectopic murine model of Lewis Lung carcinoma with a probable reduction in the toxicity of cisplatin through co-encapsulation with fisetin.

Lung cancer immunochemotherapy: Codelivery of EGFR tki and PTX via an immunostimulatory dual functional nanocarrier.

e15177 Background: Immuno-target therapy combines a target therapy agent with an immune modulating agent and represents an attractive approach to improve cancer therapy.However, the success of it is hampered by the lack of a strategy to effectively co deliver the two therapeutics to the tumours.Here we report an improved dual functional nanocarrier (PEG5k-Fmoc-NLG2) to load Gefitinib for improved cancer Immuno target therapy.PEG5K-Fmoc-NLG2 is a prodrug that can inhibited IDO enzyme activity in vitro,while IDO1 inhibition reversed T cell suppression mediated by IDO expressing mouse cancer cells. Methods: Gefitinib loaded in carrier was prepared via a film hydrationmethod,the physicochemical properties of micelles was determined by TEM and particle size test.The cytotoxicity of the drug was detected by MTT assay. 3LL lung cancer was inoculated in mice and different drugs was injected through the tail vein to compare the anti tumor effect of drug loading micells with existing targeted drug gefitinib and CTLA4 antibody; tumor tissue proteins were extracted to clarify changes in pEGFR expressionexpression; flow cytometry will be finished to observe tumor immune microenvironment changes. Results: 1. Gefitinib loaded micelle maintain the similar size with blank PEG5k-fmoc-NLG2 micelles that is around 20nm.The drug-loaded micelles produced uniformed size and morphology. which further confirmed the size of micelles was around 20nm. 2. PTX and Gefitinib loaded in PEG5k-fmoc-NLG2 exhibited significantly enhanced tumor cell killing effect than the free drug on both A549 and 3LL cell lines. 3. The antitumor efficacy of different drugs loaded in the carrier were tested in a murine lung cancer model (3LL). All treatment showed significant better antitumor activity compared to the control group, Gefitinib/PEG5k-Fmoc-NLG2 plus CTLA4 antibody exhibited the best antitumor effect. Antitumor activity of gefitinib loaded in carrier was comparable with free gefitinib alone. 4. With various treatments the expression of p-EGFR protein level varied. Basically, compare with free gefitinib alone, the carrier loaded gefitinib was more effective in the inhibition of EGFR phosphorylation. Conclusions: We have shown that targeted delivery of PTX and Gefitinib via a immunostimulatory nanocarrier effectively improved drugs antitumor activity. More study about tumor immune microenvironment are required. Such strategy can be employed in novel therapy combining chemotherapy drugs and immunemodulating agents such as PD-1 and CTLA4.

Thyroxine promotes lung cancer growth in an orthotopic mouse model.

Thyroid hormones are important for physiology and homeostasis. In addition to nuclear thyroid hormone receptors, the plasma membrane protein integrin αvβ3 has been recognized as a receptor for both thyroxine (T4) and triiodothyronine (T3). Here, we studied whether thyroid hormone promotes growth of murine lung cancer via αvβ3 in vivo. Murine Lewis lung carcinoma cells (3LL), stably transfected with luciferase, were injected into mouse lungs. Tumor growth in untreated mice was compared to hypothyroid mice and hypothyroid mice treated with T3 or T4 with or without the αvβ3 inhibitor 3,5,3',5'-tetraiodothyroacetic acid (Tetrac). Tumor progression was determined by serial in vivo imaging of bioluminescence emitted from the tumor. Tumor weight was recorded at the end of the experiment. Neoangiogenesis was determined by immunohistochemistry for CD31. Tumor growth was reduced in hypothyroidism and increased by T4 treatment. Strikingly, only T4 but not T3 treatment promoted tumor growth. This T4 effect was abrogated by the αvβ3 inhibitor Tetrac. Tumor weight and neoangiogenesis were also significantly increased only in T4-treated mice. The T4 effect on tumor weight and neoangiogenesis was abolished by Tetrac. In vitro, T4 did not stimulate 3LL cell proliferation or signaling pathway activation. We conclude that T4 promotes lung cancer growth in this orthotopic mouse model. The tumor-promoting effect is mediated via the plasma membrane integrin αvβ3 and increased neoangiogenesis rather than direct stimulation of 3LL cells. These data suggest that such effects of levothyroxine may need to be considered in cancer patients on T4 substitution.

Restoration of miR-7 expression suppresses the growth of Lewis lung cancer cells by modulating epidermal growth factor receptor signaling

microRNAs are an abundant class of short endogenous non-coding RNAs that function as important regulators of multiple target genes and participate in diverse biological roles in carcinogenesis. However, the role of miR-7 in lung cancer remains unclear and requires further elucidation. In the present study, we found a reduction of miR-7 expression in Lewis lung cancer (3LL) cells originating from mice by real-time RT-PCR. Restoration of miR-7 inhibited 3LL cell proliferation, induced cell apoptosis in vitro and reduced tumorigenicity in vivo. We further confirmed that miR-7 downregulated the expression of both epidermal growth factor receptor (EGFR) and murine leukemia viral oncogene homologue-1 (RAF-1) oncogenes by real-time PCR and western blot analysis. Furthermore, inhibition of EGFR showed similar effects to miR-7 enforcement in 3LL cells. Taken together, these findings revealed that miR-7 acts as an antitumor miRNA in 3LL by targeting and suppressing the expression of both EGFR and RAF-1 oncogenes. This study may provide a rationale for the use of miR-7 in lung cancer target therapy.

Anaphylatoxin C5a Creates a Favorable Microenvironment for Lung Cancer Progression

The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.

Nrf2-deficiency creates a responsive microenvironment for metastasis to the lung

The Nrf2 transcription factor is crucial for regulating the cellular defense against various carcinogens. However, relationship between host Nrf2 and cancer metastasis remains unexplored. To address this issue, we examined susceptibility of Nrf2-deficient mice to pulmonary cancer metastasis following implantation of the mouse Lewis lung carcinoma (3LL) cell line. Nrf2-deficient mice reproducibly exhibited a higher number of pulmonary metastatic nodules than wild-type mice did. The lung and bone marrow (BM) of cancer-bearing Nrf2-deficient mice contained increased numbers of inflammatory cells, including myeloid-derived suppressor cells (MDSCs), a potent population of immunosuppressive cells. MDSCs can attenuate CD8(+) T-cell immunity through modification of the T-cell receptor complex exploiting reactive oxygen species (ROS). MDSCs of Nrf2-deficient mice retained elevated levels of ROS relative to wild-type mice. BM transplantation experiments revealed functional disturbance in the hematopoietic and immune systems of Nrf2-deficient mice. Wild-type recipient mice with Nrf2-deficient BM cells showed increased levels of lung metastasis after cancer cell inoculation. These mice exhibited high-level accumulation of ROS in MDSCs, which showed very good coincidence to the decrease of splenic CD8(+) T-cells. In contrast, Keap1-knockdown mutant mice harboring high-level Nrf2 expression displayed increased resistance against the cancer cell metastasis to the lung, accompanied by a decrease in ROS in the MDSCs fraction. Our results thus reveal a novel function for Nrf2 in the prevention of cancer metastasis, presumably by its ability to preserve the redox balance in the hematopoietic and immune systems.

Differential Effect of Ischemia/Reperfusion Injury on the Primary and Metastatic Growth of Lewis Lung Carcinoma

Background: A growing body of evidence points toward an active role of inflammation and ischemia in tumor biology. Reperfusion injury is an inflammatory process involving natural IgM antibodies and complement activation that occurs following acute ischemia of tissues throughout the body. We hypothesized that ischemia-reperfusion (I/R) injury can affect tumor growth and spread. Methods: The Lewis Lung carcinoma (3LL) cell line was implanted by intramuscular injection into the hindlimbs of wild-type C57/BL6 or Rag2 -/- mice, which are deficient in lymphocytes and antibodies. Subsequently, tumor was subjected to varying times of tourniquet induced hind-limb ischemia, followed by tourniquet removal and reperfusion. Control mice underwent hindlimb I/R to the contralateral, non-tumor bearing hindlimb or were sham-treated. Growth of the primary tumor was followed using external caliper measurement and wet weight at sacrifice. Surface lung metastasis were counted using a stereo microscope. Tumor and surrounding hindlimb muscle were examined for histologic signs of injury. Presence of activated caspase-3 and immunoglobulin M was assessed using immunohistochemistry. Statistical analysis was performed using the Wilcoxon test. Results: I/R injury decreased 3LL lung metastasis compared to control, uninjured mice (mean number: 6.9 (sham) vs. 1.4 (I/R), p < .001). Conversely, primary tumor growth was enhanced in I/R treated animals compared to sham controls (mean weight: 9.7 g (sham) vs. 12.9 g (I/R), p < .0001). Control I/R injury to the non-tumor bearing hindlimb had no significant effect on primary (p<0.13) or metastatic (p<0.59) 3LL growth. Following I/R injury, 3LL tumors showed histologic evidence of necrosis and presence of activated caspase-3 staining, a marker of apoptosis. Deposition of IgM on I/R treated but not sham treated 3LL was seen. In 3LL implanted in Rag2 -/- mice, there was no difference in primary tumor growth in I/R injured and sham groups. However, reduction of 3LL lung metastasis following I/R treatment was preserved in Rag2 -/- mice. Conclusions: This study demonstrates that I/R injury can significantly impact tumor behavior in vivo. I/R injury inhibited 3LL lung metastasis in a lymphocyte-independent manner. In contrast, I/R injury enhanced primary 3LL tumor growth in a lymphocyte-dependent manner. These effects correlated histologically with 3LL tumor necrosis and apoptosis following I/R injury. Moreover, altered tumor behavior required the direct, local effect of I/R injury and not its systemic effects alone. Further studies are required to delineate the pathways underlying the differential impact of I/R injury on primary and metastatic tumor growth.

Establishment of orthotopic Lewis lung cancer model in mouse

背景与目的肺癌原位模型包括小鼠自发性肺肿瘤模型和气道内接种模型等，但自发性肺肿瘤模型耗时较长且成瘤率不能保证，而气道内接种模型成瘤部位及大小不稳定。本研究以3LL细胞系细胞接种于C57BL/6小鼠肺原位，与皮下接种模型比较，探讨其稳定性、转移特性，并建立小鼠肺原位癌模型的优化方法。方法将不同数量级的3LL细胞分别直接接种于C57BL/6小鼠左侧腋下制备皮下接种模型和以Matrigel悬浮后接种于其左肺制备原位接种模型，观察两种模型的生存期，并对小鼠进行解剖后行病理切片检查、免疫组化染色检测微血管密度、流式细胞仪检测CD44v。结果皮下组成瘤率分别为100%、66.7%、16.7%，未见明显转移。原位组成瘤率分别为100%、100%、83.3%，并可转移至对侧胸廓及肺脏。原位组中位生存期（38 d、35 d、23 d）明显少于皮下接种组（82 d、72 d、50 d）。原位接种组微血管密度（120.2±9.73）高于皮下组（92.6±7.12）。原位接种组肿瘤细胞悬液CD44v表达（26.46±1.56）%高于皮下接种组（23.13±1.02）%。结论以3LL细胞接种于小鼠肺部所建立的肺癌原位模型简单可靠，重复性高，具有较皮下接种模型更强的转移特性。

IFN-γ Acts on T Cells to Induce NK Cell Mobilization and Accumulation in Target Organs

The mechanism(s) that regulates NK cell mobilization and the significance of this process to NK cell activity are unknown. After Con A-induced hepatitis, NK cells are mobilized from the spleen and bone marrow into the periphery in an IFN-gamma-dependent fashion. Intraperitoneal administration of IFN-gamma stimulates the mobilization of NK cells into the circulation, but not their cell death or proliferation. Increased number of circulating NK cells was coupled with their accumulation in the peritoneum, liver, and tumor-bearing lung tissue. Furthermore, increased number of NK cells in the lung reduced metastasis of Lewis lung carcinoma cells (3LL cell line) resulting in significantly extended NK-dependent survival. Mobilization of NK cells was specific and required the presence of T cells. Moreover, mobilization and migration of spleen NK cells in response to IFN-gamma treatment is dependent on the chemokine receptor CXCR3. Mechanistic insights regarding the role of IFN-gamma in the regulation of NK cell mobilization and their accumulation at sites of tumor metastasis may lead to the development of novel immunotherapy for cancer.

Coming Back at You with PI3K

The treatment of many solid cancers involves surgical removal of the primary tumor, a process that, dismayingly, may itself potentiate the growth of residual disease. Coffey et al. developed a model system in which they generated flank tumors in mice and measured the growth of primary tumors and recurrent tumors after surgical removal of most of the tumor. In immunocompromised mice with tumors of human origin and non-immunocompromised mice with tumors derived from the murine 3LL cell line, recurrent tumors grew more rapidly than primary tumors. Apoptosis (determined by morphological changes and TUNEL immunohistochemistry or by propidium iodide uptake) was reduced in recurrent compared with primary tumors, and the growth and metastatic capability of cells isolated from recurrent tumors was greater than that of cells isolated from primary tumors. Moreover, epithelial cells isolated from recurrent tumors showed greater resistance to tumor necrosis factor apoptosis-inducing ligand (TRAIL). Gene expression profiling indicated that expression of the gene encoding the phosphoinositide 3-kinase (PI3K) p85α inhibitory subunit ( Pik3r1 ) was decreased in recurrent tumors when compared with its expression in primary tumors. However, expression of the gene encoding the PI3K p110γ catalytic subunit ( Pik3CA ) was increased in recurrent tumors. Consistent with a shift in PI3K activity, phosphorylation of Akt was increased in recurrent tumors. LY294002, an inhibitor of PI3K, increased the susceptibility of recurrent tumor cells to TRAIL without affecting that of primary tumor cells and, when administered postoperatively, slowed recurrent tumor growth and prolonged survival. Thus, accelerated growth of tumors following surgery may involve increased PI3K activity, and inhibition of PI3K activity in the immediate postoperative period could potentially mitigate this effect. J. C. Coffey, J. H. Wang, M. J. F. Smith, A. Laing, D. Bouchier-Hayes, T. G. Cotter, H. P. Redmond, Phosphoinositide 3-kinase accelerates postoperative tumor growth by inhibiting apoptosis and enhancing resistance to chemotherapy-induced apoptosis: Novel role for an old enemy. J. Biol. Chem. 280 , 20968-20977 (2005). [Abstract] [Full Text]

729. C-Met Antisense Plasmid Liposome Complex Increases Radiation Sensitivity of Lung Tumor Cells In Vivo or In Vitro

Lung cancer cells have an increased expression of hepatocyte growth factor (HGF) receptor. Stimulation of the HGF receptor may be responsible for the rapid growth of lung tumors. Transfection of lung tumor cells with a plasmid containing the c-Met antisense transgene decreases expression of hepatocyte growth factor (HGF) receptor and increases apoptosis. To determine whether the increased expression of the c-Met antisense sensitize cells to irradiation, three lung tumor cell lines (3LL murine cell line, 201T human cell line, and A549 human cell line) were transfected with either a control plasmid or a plasmid containing the c-Met antisense transgene. Irradiation survival curves were performed 24 hours later. Seven days later colonies were stained with crystal violet and colonies of greater than 50 cells were counted. There was no statistical difference in the irradiation survival curves for the control cells compared to the cells transfected with the control plasmid by the Do or the shoulder on the survival curve (n). In contrast in both A549 and 201T cell lines transfected with the c-Met antisense plasmid there was an increased irradiation sensitivity as seen by a decreased shoulder on the survival curve compared to the non-transfected irradiated cells (n = 3.05 |[plusmn]| 0.45 compared to 6.43 |[plusmn]| 1.03 and 3.00 |[plusmn]| 1.31 compared to 5.95 |[plusmn]| 0.21, respectively). Cells from the 3LL cell line transfected with the c-Met antisense plasmid demonstrated radiation sensitivity by a decreased Do compared to the control irradiated but nontransfected cells (1.40 |[plusmn]| 0.18 Gy compared to 1.73 |[plusmn]| 0.09 Gy, respectively). In a separate experiment, cells were grown on cover slips, irradiated to 10 Gy and 48 hours later, the percent apoptotic cells were determined using a tunnel assay. For the 3LL, A549 and 201T cell lines there was increased apoptosis following c-Met antisense transfection of the tumor cells (9.3 |[plusmn]| 0.9%, 68.1 |[plusmn]| 2.0%, 4.6 |[plusmn]| 0.5%, respectively, compared to 0% in the control nonirradiated cells). There was an additional increase in the percent of apoptotic cells following irradiation in the c-Met antisense transfected 3LL, A549, and 201T cells compared to the irradiated control non-transfected cells (91.6 |[plusmn]| 1.4%, 94.2 |[plusmn]| 0.8%, or 95.9 |[plusmn]| 1.1%, respectively, compared to 0% in the control irradiated cells). To determine the effect of c-Met antisense gene therapy in the in vivo orthotopic tumor model, C57BL/6J mice were injected intratracheally with 3LL lung tumor cells. Beginning one day later and continuing for the next three days, the mice were injected I.V. with c-Met antisense plasmid liposome complexes. The mice were irradiated 48 hours after tumor implantation to 18 Gy to the pulmonary cavity. Mice injected with the antisense c-Met plus 18 Gy had improved survival compared to mice injected with the 3LL cells alone plus 18 Gy of irradiation. Optimization of the delivery of the c-Met antisense plasmid liposome complexes may facilitate radiosensitization of lung cancer.

1069. Increased Antioxidant Levels Following MnSOD-PL Transfection Results in Increased Radioprotection of Normal Tissue but Not Tumor Cells

Top of pageAbstract Administration of Manganese Superoxide Dismutase Plasmid/Liposome complexes (MnSOD-PL) to the lung, esophagus, oral cavity, intestine and bladder has resulted in increased radioprotection of normal tissue. However, transfection of orthotopic tumors with MnSOD-PL has resulted in sensitization of the tumors to irradiation. One hypothesis is that the increased MnSOD expression produces increased hydrogen peroxide. Tumor cells may have a decreased ability to reduce hydrogen peroxide due to decreased antioxidant levels resulting in increased concentrations of hydrogen peroxide, leading to cell toxicity. Normal cells with higher levels of antioxidants may be able to reduce hydrogen peroxide and prevent cell death. To test this hypothesis, we measured antioxidant levels in a normal embryonic fibroblast cell and three tumor cell lines (3LL murine lung tumor, SSC-VII murine oral cavity cancer, or OC-19 human oral cavity cancer). Cells from each of the cell lines were transfected with a plasmid containing the human MnSOD transgene. Twenty-four hours later, the transfected cells as well as non-transfected cells were irradiated to 10 Gy. Irradiated cells as well as non-irradiated cells were harvested after 30 mins., and glutathione (GSH) or glutathione peroxidase (GPX) activities measured. All four cell lines following transfection with the MnSOD plasmid had significant increases in GSH levels. However, after irradiation, OC-19 and SCC-VII cells had significantly decreased GSH compared to GSH levels (65 |[plusmn]| 0.6 decreased to 43.6 |[plusmn]| 0.7 |[mu]|M, and 29.8 |[plusmn]| 1.9 decreased to 20.2 |[plusmn]| 1.4 |[mu]|M, respectively). In contrast the normal fibroblast cell line and the 3LL cell line showed no irradiation induced change in the level of GSH. GPX activity decreased following irradiation in all four nontransfected cell lines. Following transfection with the MnSOD plasmid there was significantly decreased GPX activity following irradiation in the 3LL, SCC-VII, and OC-19 lines (56.3 |[plusmn]| 2.5 to 49.0 |[plusmn]| 1.6, 66.1 |[plusmn]| 1.3 to 37.5 |[plusmn]| 1.1, 45.0 |[plusmn]| 1.6 to 37.1 |[plusmn]| 0.9 mU/mg protein, respectively). However, in the normal fibroblast cell line, there was no change in GPX activity following irradiation in transfection or the non-transfected cells (22.1 |[plusmn]| 3.1 and 21.5 |[plusmn]| 1.9, respectively). In vivo analysis of orthotopic SSC-VII tumors in the oral cavity of C3H/HeNHsd mice transfected by intravenous injections of MnSOD plasmid/liposome complexes showed decreases in total thiols following irradiation compared to nonirradiated orthotopic tumors (56.8 |[plusmn]| 1.1, and 33.9 |[plusmn]| 0.9 nmol/mg protein, respectively). Irradiation did not cause a decrease in protein SH groups in nontransfected SSC-VII tumors (56.8 |[plusmn]| 1.1 and 60.7 |[plusmn]| 6.4 nmol/mg protein, respectively). These data demonstrate that transfection with MnSOD-PL carries an irradiation induced decrease in antioxidants in tumor cells. Reduction in the antioxidant pools may increase the radiosensitivity of tumors.

C3‐induced 3LL cell proliferation is mediated by C kinase

It has been demonstrated that the third component of complement (C3)(1) and its peptides increase normal and tumour cell proliferation. However, the signal cascade responsible for this phenomenon is still unknown. In this study, we elucidate some of the mechanisms involved in the signalling of C3 stimulation of cell proliferation. We have first investigated the in and out traffic of C3 peptides, then we have identified the subcellular localisation of internalised C3 and, finally, we have explored the role of protein phosphorylation in C3 traffic and in the proliferation of the Lewis lung carcinoma (3LL) cells. Our results indicate that traffic of C3 is not dependent on cytoskeletal integrity and requires protein kinase C-dependent phosphorylation. In addition, proliferation of 3LL cells stimulated by C3 depends on both C3 internalisation and protein-kinase C phosphorylation.

Effect of procyanidin on cell cycle in murine lewis lung carcinoma

Objective: To investigate the anti-tumor effect of procyanidin (WeiMaiNing, WMN) on a murine Lewis lung carcinoma cell line (3LL) and the influence on the cell cycle. Methods: The inhibitory rate of 3LL growth was detected in the model of murine Lewis lung carcinoma. The effect of the drug on 3LL cell cycle and the influence of the drug on the expression of cyclin D1 protein were investigated by flow cytometry, immunohistochemical staining. Results: The inhibitory rates of WMN in 3LL were 19.14%, 33.59% and 51.56% respectively at dosages 100 mg·kg−1·d−1, 150 mg·kg−1·−1 and 250 mg·kg−1·d−1. The inhibitory effect was in a dose-dependent manner. We found WMN significantly increased (P<0.01) the number of 3LL cells in G0–G1 phase (35.97% vs. 27.2% at 150 mg·kg−1·−1 WMN; 40.10% vs. 27.2% at 250 mg·kg−1·−1 WMN) and decreased the expression of cyclin D1, PCNA protein. Conclusion: WeiMaiNing inhibits the growth of 3LL cells in vivo by decreasing the expression of PCNA and cyclin D1, blocking the cells in G0–G1 phase and preventing the cells transition from G1 to S phase while DNA is replicated.

Endocytosis of VEGFR-2 on endothelial progenitor cells by dopamine attenuates tumour growth and vasculogenesis

Introduction: Endothelial progenitor cells (EPC’s) andvasculogenesis are intricately involved in the initial neovascularisation of tumours. EPC’s are characterised by the expression of VEGFR-2 and AC133. Vasculogenesis offers a novel possibility for the targeting of anti-cancer therapies. Methods: Ex-vivo expanded EPC’s were cultured from peripheral blood samples from healthy male volunteers. To endocytose VEFGR-2 on EPC’s, cells were treated with incremental doses of dopamine and VEGFR-2 expression was analysed using anti-VEGFR-2 mAb on flow cytometry. Dil-labelled ex-vivo expanded EPC’s were transplanted into tumour bearing (3LL tumour cell line) MF1-nude mice (n=12). The mice received a daily i.p. injection of PBS or Dopamine (50 mg/kg). Tumour volume was assessed on alternate days. Vasculogenesis and angiogenesis were assessed using fluorescent microscopy and CD31 staining. To determine if Dopamine has a direct effect on 3LL cells, cells were cultured with incremental doses of dopamine for 48 h and cell apoptosis (PI-staining), proliferation (BrdU-Assay) and Cytotoxicity (LDH-Assay) were analysed. Statistical analysis was performed on SigmaStat using ANOVA. Results: Ex-vivo expanded EPC cultures were 80–85% pure. Dopamine caused an endocytosis of VEGFR-2 on EPC’s with the maximal effect seen at 1μM (p < 0.05). Dopamine had no effect on 3LL cell apoptosis, proliferation or cytotoxicity in all doses performed. Dopamine, by endocytosing VEGFR-2 on EPC’s, attenuated tumour growth by decreasing both tumour volume (p < 0.05; 241.8 +/- 106.8 v’s 59.7 +/- 20.5 at day 10) and tumour weight (p < 0.05). Absolute and relative levels of vasculogenesis were decreased in the dopamine group as compared to controls (p < 0.05). Conclusion: Induction of VEGFR-2 endocytosis on EPC’s decreases tumour growth and vasculogenesis. This data suggests that VEGFR-2 is a target for attenuating tumour vasculogenesis.

Decrease of metastatic ability after selection for intravasating ability in Lewis lung carcinoma (3LL) cell line

The release of cancer cells from the primary site and penetration into blood vessels are obligatory preliminary steps for metastasis. To investigate the mechanism of such steps we isolated variant cells (designated as Int-3LL) possessing enhanced intravasating ability from Lewis lung carcinoma (3LL) cells by in vivo selection. In spite of the enhanced intravasating ability of Int-3LL, the spontaneous and experimental metastatic abilities of Int-3LL decreased significantly compared to parent cells. Such a cell line has never been reported so far. The matched pair of cell lines described in this report provides a useful system for investigating the primary steps of metastasis.

Protective Effect of NK1.1+T Cells as Well as NK Cells against Intraperitoneal Tumors in Mice

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1+αβT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity.In vivodepletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1+T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1.1+T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4+T cells and Vβ8+T cells compared with liver NK1.1+T cells and normally lack Vβ2+T cells. Tumor inoculation induced rapid IL-12 and IFN-γ mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatmentin vivoabrogated IFN-γ mRNA expression and IFN-γ production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-γ mRNA expression and partly inhibited IFN-γ production of TIM. Peritoneal NK cells as well as NK1.1+T cells but not NK1.1−T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-γ Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1+T cells to produce IFN-γ and both NK cells and NK1.1+T cells are important in suppressing the growth of the intraperitoneal tumors.

Echistatin inhibits Lewis lung carcinoma cell-matrix adhesion in vitro and experimental metastasis in vivo

Echistatin, a low molecular weight, RGD-containing protein isolated from the venom of Echis carinatus, inhibited Lewis lung carcinoma cell (3LL) adhesion to immobilized fibronectin and laminin. The inhibition was specific, noncytotoxic, dose-dependent and fully reversible. Echistatin showed a stronger activity in inhibiting cell adhesion to fibronectin rather than to laminin and it resulted about 3-fold more effective than kistrin, an other ROD-snake venom protein, in inhibiting 3LL cell attachment to both substrates. The ability of echistatin to modulate experimental metastasis formation in vivo was also evaluated. A 20% inhibition of the lung metastasis spread with respect to controls was observed when 3LL cells and echistatin were coinjected i.m. into male C57BL/6NCr1BR mice. When echistatin was administered i.p. 1 mu g/g of body weight/72 h x 4 doses into mice bearing Lewis lung carcinoma, it promoted only a 15% inhibition of tumor growth but inhibited by 45% lung metastasis formation. These results demonstrate that echistatin is able to inhibit metastasis attachment and spreading in experimental system in vivo independently by its effect on the primary tumor.

Liver NK1.1+ CD4+ alpha beta T cells activated by IL-12 as a major effector in inhibition of experimental tumor metastasis.

We demonstrate herein evidence that IL-12-activated alpha beta T cells with intermediate TCR (NK1+ TCRint cells) in the liver inhibit metastases in the lung as well as in the liver metastases of i-v. injected tumors. IL-12 administration enhanced NK1 expression of NK1+ TCRint cells (NK1high) and increased CD4 weakly positive (CD4low) TCRint cells, while both CD4+ TCRint cells and double-negative TCRint cells were proportionally diminished. Accordingly, the major parts of NK1high TCRint cells are CD4low cells, and most of these cells are V beta 8+ cells. The cytotoxic assays of IL-12-stimulated hepatic mononuclear cells after treatment with respective Abs and complement in vitro and after sorting revealed that CD4low NK1high TCRint cells are cytotoxic effectors. When IL-12-stimulated hepatic mononuclear cells (but not splenocytes) were transferred into tumor-preinjected mice, EL-4 cell metastases in the liver as well as 3LL cell metastases in the lung were inhibited. The antimetastasis of hepatic mononuclear cells transfer was abrogated by the depletion of NK1+ cells, CD3+ cells, or CD4+ cells but not CD8+ cells before transfer. Moreover, transfer of these cells of nude mice into tumor-preinjected mice also inhibited metastases in both organs. Although NK1+ TCRint cells are nearly absent in the hepatic vein blood, a significant proportion of NK1high TCRint cells appeared by IL-12 administration. These results demonstrate that IL-12-stimulated liver NK1high TCRint cells, including extrathymic ones, are major effectors against tumor metastasis and suggest that the cells migrate and inhibit lung metastases.

12-Lipoxygenase in Lewis lung carcinoma cells: Molecular identity, intracellular distribution of activity and protein, and Ca 2+-dependent translocation from cytosol to membranes

Recently we demonstrated that Lewis lung (3LL) tumor cells express 12-lipoxygenase (12-LOX) mRNA and protein, respectively. In this study we partially sequenced the 12-LOX cDNA after reverse-transcription polymerase chain reaction amplification of 12-LOX mRNA from cultured 3LL cells. Comparison with platelet and leukocyte 12-LOX indicates that 3LL 12-LOX is identical with the platelet-type enzyme at least within the sequenced region. Further, we investigated the intracellular distribution of both 12-LOX enzyme protein and its activity which are prerequisites for understanding 12-LOX regulation. 12-LOX activity was monitored via the production of 12-hyroxyeicosatetraenoic acid from 3LL cells and their subcellular fractions using reverse-phase high performance liquid chromatography. 12-LOX protein was measured by direct slot blot and by Western Blotting. In 3LL cells, both 12-LOX activity and 12-LOX protein were predominantly localized in the cytosol. This 12-LOX activity was optimal at 37°C. However at 24°C and 10°C, it showed 87% and 61% of this activity, respectively, thus differing distinctly from 12-LOX in platelets or rat basophilic leukemia cells. Incubation of 3LL cell homogenates with 0–100 μM free Ca 2+ and subsequent separate analyses of cytosol and membrane fractions indicated that, as in platelets, an increase in intracellular free Ca 2+ caused a loss of cytosolic 12-LOX activity. However, no significant Ca 2+-induced increase in membrane-associated 12-LOX activity was observed under these conditions in 3LL cells. In contrast, at the 12-LOX protein level we observed a Ca 2+-dependent loss in the cytosol and a concomitant increase in the membrane fraction. Thus, we suggest that 12-LOX in 3LL cells undergoes rapid translocation from cytosol to membrane in a Ca 2+-dependent manner, but is no longer active or becomes inactivated at the membrane site.