WEHI-164 Fibrosarcoma Research Articles

Comparative effects of free doxorubicin, liposome encapsulated doxorubicin and liposome co-encapsulated alendronate and doxorubicin (PLAD) on the tumor immunologic milieu in a mouse fibrosarcoma model.

Background: We have previously shown that alendronate, an amino-bisphosphonate, when reformulated in liposomes, can significantly enhance the efficacy of cytotoxic chemotherapies and help remodel the immunosuppressive tumor microenvironment towards an immune-permissive milieu resulting in increased anticancer efficacy. In addition, we have previously shown that the strong metal-chelating properties of alendronate can be exploited for nuclear imaging of liposomal biodistribution. To further improve anticancer efficacy, a pegylated liposome formulation co-encapsulating alendronate and doxorubicin (PLAD) has been developed. In this study, we examined the effects of PLAD on the tumor immunologic milieu in a mouse fibrosarcoma model in which the tumor microenvironment is heavily infiltrated with tumor-associated macrophages (TAM) that are associated with poor prognosis and treatment resistance.Methods: Doxorubicin biodistribution, characterization of the tumor immunologic milieu, cellular doxorubicin uptake, and tumor growth studies were performed in Balb/c mice bearing subcutaneously implanted WEHI-164 fibrosarcoma cells treated intravenously with PLAD, pegylated liposomal doxorubicin (PLD), free doxorubicin, or vehicle.Results: PLAD delivery resulted in a high level of tumor doxorubicin that was 20 to 30-fold greater than in free doxorubicin treated mice, and non-significantly higher than in PLD treated mice. PLAD also resulted in increased uptake in spleen and slightly lower plasma levels as compared to PLD. Importantly, our results showed that PLAD, and to a lesser extent PLD, shifted cellular drug uptake to TAM and to monocytic myeloid-derived suppressor cells (MDSC), while there was no drug uptake in neutrophilic MDSC or lymphoid cells. Free doxorubicin cellular drug uptake was below detectable levels. PLAD, and to a lesser extent PLD, also induced significant changes in number and functionality of tumor-infiltrating TAM, MDSC, Treg, NKT, and NK cells that are consistent with enhanced antitumor immune responses in the tumor microenvironment. In contrast, free doxorubicin induced moderate changes in the tumor microenvironment that could promote (decreased Treg) or be detrimental to antitumor immune responses (decreased M1 TAM and NK cells). These immune modulatory effects are reflected in the therapeutic study which showed that PLAD and PLD inhibited tumor growth and significantly prolonged survival, while free doxorubicin showed little or no anticancer activity.Conclusion: We show that liposomal delivery of doxorubicin not only alters pharmacokinetics, but also dramatically changes the immune modulatory activity of the drug cargo. In addition, our data support that the PLAD nanotheranostic platform further enhances some immune changes that may act in synergy with its cytotoxic chemotherapy effects.

The characterization of exosomes from fibrosarcoma cell and the useful usage of Dynamic Light Scattering (DLS) for their evaluation.

Exosomes are a type of extracellular vesicles containing mRNA, miRNA, and proteins of origin cells, which can control the characteristics of other cells or surroundings. Despite increasing evidence on oncogenic properties of tumor-derived exosomes, fibrosarcoma-derived exosomes remain largely unrevealed. While the proper extraction and characterization of exosomes is critical in exosomes research, there are various limitations in techniques to measure the size and homogeneity of exosomes. Here, we analyzed exosomes from a fibrosarcoma cell line WEHI-164 compared with a breast cancer cell line MDA-MD-231 as a control. Results from dot blot and western blot analysis demonstrated that GM1 ganglioside, and TSG101, HSC70 and GAPDH proteins were contained in exosomes from the WEHI-164 fibrosarcoma cell line. The existence of tetraspanins such as CD81, CD63 and CD9 was confirmed in the exosomes by ExoView analysis. The results obtained from TEM showed their sphere-like shapes of around 50 to 70 nm in radius. Through DLS, we found out that the mean radius of the exosomes derived from WEHI-164 and MDA-MB-231 cell lines was 94.4 nm and 107.8 nm, respectively, with high homogeneity. When comparing the radius measured by TEM with the radius measured by DLS, it was revealed that the difference between the two methods was about 40 nm. This study has significance in characterizing the molecular properties of exosomes from a fibrosarcoma, which has not been researched much before, and in providing clear evidence that DLS can be used as an efficient, convenient and noninvasive technique to simply check the homogeneity and size of exosomes.

Optimized Dose of Dendritic Cell-based Vaccination in Experimental Model of Tumor Using Artificial Neural Network.

Previous studies have demonstrated that maturation of dendritic cells (DCs) by pathogenic components through pathogen-associated molecular patterns (PAMPs) such as Listeria monocytogenes lysate (LML) or CpG DNA can improve cancer vaccination in experimental models. In this study, a mathematical model based on an artificial neural network (ANN) was used to predict several patterns and dosage of matured DC administration for improved vaccination. The ANN model predicted that repeated co-injection of tumor antigen (TA)-loaded DCs matured with CpG (CpG-DC) and LML (List-DC) results in improved antitumor immune response as well as a reduction of immunosuppression in the tumor microenvironment. In the present study, we evaluated the ANN prediction accuracy about DC-based cancer vaccines pattern in the treatment of Wehi164 fibrosarcoma cancer-bearing mice. Our results showed that the administration of the DC vaccine according to ANN predicted pattern, leads to a decrease in the rate of tumor growth and size and augments CTL effector function. Furthermore, gene expression analysis confirmed an augmented immune response in the tumor microenvironment. Experimentations justified the validity of the ANN model forecast in the tumor growth and novel optimal dosage that led to more effective treatment.

Sarcoma Eradication by Doxorubicin and Targeted TNF Relies upon CD8+ T-cell Recognition of a Retroviral Antigen.

Antibody-cytokine complexes may offer new tools to treat cancer. Here, we show how TNF-linked antibodies, which recognize tumor-selective splice isoforms of fibronectin (F8-TNF), can be exploited to eradicate sarcomas in immunocompetent mice. We treated mice bearing WEHI-164 fibrosarcoma with a combination of F8-TNF and doxorubicin, curing the majority of treated animals (29/37). Notably, cured mice were resistant to rechallenge not only by WEHI-164 cells but also heterologous C51 or CT26 colorectal tumor cells in a CD8+ T-cell-dependent process. Mechanistic analyses revealed that each tumor cell line presented AH1, a common endogenous retroviral peptide. Numbers of AH1-specific CD8+ T cells exhibiting cytotoxic capacity were increased by F8-TNF plus doxorubicin treatment, arguing that cognate CD8+ T cells contributed to tumor eradication. Sequence analysis of T-cell receptors of CD8+ T cells revealed the presence of H-2Ld/AH1-specific T cells and an expansion of sequence diversity in treated mice. Overall, our findings provide evidence that retroviral genes contribute to tumoral immunosurveillance in a process that can be generally boosted by F8-TNF and doxorubicin treatment. Cancer Res; 77(13); 3644-54. ©2017 AACR.

The therapeutic T‐cell response induced by tumor delivery of TNF and melphalan is dependent on early triggering of natural killer and dendritic cells

The fusion protein L19mTNF (mouse TNF and human antibody fragment L19 directed to fibronectin extra domain B) selectively targets the tumor vasculature, and in combination with melphalan induces a long-lasting T-cell therapeutic response and immune memory in murine models. Increasing evidence suggests that natural killer (NK) cells act to promote effective T-cell-based antitumor responses. We have analyzed the role of NK cells and dendritic cells (DCs) on two different murine tumor models: WEHI-164 fibrosarcoma and C51 colon carcinoma, in which the combined treatment induces high and low rejection rates, respectively. In vivo NK-cell depletion strongly reduced the rejection of WEHI-164 fibrosarcoma and correlated with a decrease in mature DCs, CD4+ , and CD8+ T cells in the tumor-draining LNs and mature DCs and CD4+ T cells in the tumor 40 h after initiation of the therapy. NK-cell depletion also resulted in the impairment of the stimulatory capability of DCs derived from tumor-draining LNs of WEHI-164-treated mice. Moreover, a significant reduction of M2-type infiltrating macrophages was detected in both tumors undergoing therapy. These results suggest that the efficacy of L19mTNF/melphalan therapy is strongly related to the early activation of NK cells and DCs, which are necessary for an effective T-cell response.

Applications of calcium electroporation to effective apoptosis induction in fibrosarcoma cells and stimulation of normal muscle cells

The electroporation (EP) supports various types of anticancer therapies by the selective transport of cytostatics. Increase in intracellular calcium level by EP may be a new approach to fibrosarcoma treatment. Calcium is one of the most important factors of cell proliferation, differentiation and cell death (apoptosis or necrosis). Calcium level balanced by electroporation can cause different effects on normal and pathological cells. The efficiency and safety of electroporation combined with Ca2+ ions were examined in our study. The two muscle cell lines were used: normal rat skeletal muscle cells — L6 and cancer muscle cells — Wehi-164 (fibrosarcoma). Two CaCl2 concentrations were tested: 0.5mM and 5mM combined with EP parameters: 1000V/cm, 1200V/cm, and 1500V/cm. The results show that EP supported by Ca2+ is cytotoxic for Wehi-164 cells and simultaneously safe for normal muscle cells. The main type of cell death – apoptosis – was confirmed by Tunnel and Annexin V/PI assay. Additionally, sPLA2 pro-tumorigenic influence was proved by immunocytochemistry. Moreover, EP with 0.5mM of Ca2+ slightly stimulates the normal muscle cells — L6 to increase proliferation.

Immunomodulatory effect of β-glucan on peritoneal macrophages of Babl/c mice

We assessed the effect of β-Glucan on macrophages by Griess reagent and viability by MTT assay and cytotoxicity. Assay of macrophages culture supernatants were carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay were done. NO release was increased at the dose of 10 µg/ml (P = 0.001) of β-Glucan while the viability of macrophages in all concentrations was the same. In TNF-α bioassay, the supernatant of macrophages stimulated with β-Glucan had a significant cytotoxic effect on WEHI-164 cells (P = 0.023). β-Glucan had a positive effect on increasing tumoricidal activity of macrophages which may help in anti-cancer immune responses.

Methanolic Fractions of Ornithogalum cuspidatum Induce Apoptosis in PC-3 Prostate Cancer Cell Line and WEHI-164 Fibrosarcoma Cancer Cell Line.

The present study, was aimed to assess the cytotoxic effects of Ornithogalum cuspidatum methanolic fractions on PC-3, prostate cancer cells and WEHI-164, Fibrosarcoma cells. Methanolic fractions of O. cuspidatum were prepared using solid phase extraction and the cells were treated with different concentrations for 12 and 24 hours. Cytotoxicity and cell viability were measured by MTT assay. ELISA was also employed to assess the histone-associated DNA fragments and the involvement of apoptotic mechanisms. 10 and 20% fractions had not significant cytotoxic effects (p>0.05) but other fractions exerted growth inhibition on both cancer cell lines (p<0.05). After 24h of incubation with 40, 60, 80 and 100% fractions, the IC50 values were: 165, 85, 65 and 45μg/ml on PC-3 cells and 200, 96, 76 and 73μg/ml against WEHI-164 cell line, respectively. ELISA results also revealed that, both cell lines had undergone apoptosis. It is deduced that, 80% and 100% methanolic fractions had significant anti-proliferative and apoptotic impacts on PC-3 and WEHI-164 cells in vitro and could be considered for developing chemo-preventive substances.

Evaluation of anti-cancer and immunomodulatory effects of carnosol in a Balb/c WEHI-164 fibrosarcoma model

Agents that destroy tumor cells and simultaneously boost host anti-tumor immunity are of keen interest in cancer therapy. In the present study, the effect of carnosol on anti-tumor immunity in a Balb/c mouse model of fibrosarcoma was evaluated. Carnosol was administered intraperitoneally daily (at 5 or 10 mg/kg/day, for 7 days) to tumor-bearing mice (i.e. 7 days after initial injection of tumor cells). Another group of tumor-bearing mice was treated with 20 mg cyclophosphamide/kg/d (positive control); a final group received vehicle only (vehicle control). After an initial measure on Day 0, tumor size was measured twice during the 7-day treatment period. One day after the final treatment with vehicle/carnosol (i.e. Day 7), the mice had their tumors measured and then were euthanized to permit their spleen and tumor to be harvested for isolation of, respectively, splenocytes and tumor-associated lymphocytes. Using these materials, spontaneous and mitogen-induced release of interleukin (IL)-4, IL-10, and interferon (IFN)-γ, lymphocyte proliferation, and the absolute numbers/relative percentages of splenic and tumor-associated T-regulatory (Treg) and other T-lymphocyte sub-sets were evaluated. The results showed that carnosol at both doses significantly suppressed tumor growth and caused depletion of splenic and tumor-associated Treg cells. It also caused relative (vs control mouse cell values) decreases in splenocyte spontaneous/inducible production of IL-4 and IL-10 and increases in IFNγ and cell proliferation. Carnosol at either dose did not cause changes in the percentages of CD4+ or CD8+ lymphocytes in the spleen or in tumor-associated lymphocyte populations. The observed increases in IFNγ, decreases in IL-10 and IL-4 production, and reductions in splenic/tumor-associated Treg cell levels might be signs reflecting the potential anti-tumor activity of carnosol. Based on the findings here, it is asserted that carnosol is a likely candidate – after more complete toxicologic evaluation – for eventual use as an anti-cancer therapeutic.

The dose-dependent tumor targeting of antibody-IFNγ fusion proteins reveals an unexpected receptor-trapping mechanism in vivo.

Cytokines often display substantial toxicities at low concentrations, preventing their escalation for therapeutic treatment of cancer. Fusion proteins comprising cytokines and recombinant antibodies may improve the anticancer activity of proinflammatory cytokines. Murine IFNγ was appended in the diabody format at the C-terminus of the F8 antibody, generating the F8-IFNγ fusion protein. The F8 antibody is specific for the extra-domain A (EDA) of fibronectin, a tumor-associated antigen that is expressed in the vasculature and stroma of almost all tumor types. Tumor-targeting properties were measured in vivo using a radioiodinated preparation of the fusion protein. Therapy experiments were performed in three syngeneic murine models of cancer [F9 teratocarcinoma, WEHI-164 fibrosarcoma, and Lewis lung carcinoma (LLC)]. F8-IFNγ retained the biologic activity of both the antibody and the cytokine moiety in vitro, but, unlike the parental F8 antibody, it did not preferentially localize to the tumors in vivo. However, when unlabeled F8-IFNγ was administered before radioiodinated F8-IFNγ, a selective accumulation at the tumor site was observed. F8-IFNγ showed dose-dependent anticancer activity with a clear superiority over untargeted recombinant IFNγ. The anticancer activity was potentiated by combining with F8-IL4 without additional toxicities, whereas combination of F8-IFNγ with F8-TNF was lethal in all mice. Unlike other antibody-cytokine fusions, the use of IFNγ as payload for anticancer therapy is associated with a receptor-trapping mechanism, which can be overcome by the administration of a sufficiently large amount of the fusion protein without any detectable toxicity at the doses used.

Soy protein and genistein improves renal antioxidant status in experimental nephrotic syndrome.

Nephrotic syndrome is a chronic disease especially common in the childhood and adolescence. Reactive oxygen species (ROS) and free radicals have significant role in the pathogenesis of nephrotic syndrome. The aim of this study was to evaluate the effect of soy protein and genistein (main isoflavone of soybean) on renal antioxidant status of nephrotic rats. This study was done for 8 weeks on 40 adult male Sprague-Dawley rats divided into four groups of 10 rats each. Study groups included: 1-Control, 2-Nephrotic syndrome, 3-Nephrotic syndrome+soy protein diet and 4-Nephrotic syndrome+soy protein diet+genistein. Urine protein and urine creatinine were measured. After homogenization of kidney, total antioxidant capacities (TAC), activities of catalase enzyme, the concentration of malondialdehydes (MDA) and carbolynated proteins were determined spectrophotometrically. Pathological examination was done on kidneys with light microscope. Cell viability was evaluated with MTT assay on WEHI-164 fibro sarcoma cell line. The MMP2 enzyme activity was evaluated in different concentrations of genistein. Total antioxidant capacity was significantly increased in soy genistein. Catalase activity was significantly increased in soy and soy genistein groups. Protein carbonyl and MDA were significantly lower in soy and soy genistein groups. The scores of pathological examination showed significant improvement in soy and soy genistein groups. Genistein decreased the proliferation of the WEHI-164 fibrosarcoma cell line. It seems that soy protein decreases kidney damages in nephrotic syndrome. Adding genistein to soy protein causes improvements in antioxidant status of kidney tissue. Genistein decreases proliferation of cell.

Effect of aged garlic extract on immune responses to experimental fibrosarcoma tumor in BALB/c mice.

Aged garlic extract (AGE) has many biological activities including radical scavenging, antioxidative and immunomodulative effects. In this research work, the antitumor and immunomodulatory effects of AGE against fibrosarcoma implanted tumor were studied. WEHI-164 fibrosarcoma cells were implanted subcutaneously on day 0 into the right flank of 40 BALB/c mice at age of 8 weeks. Mice were randomly categorized in two separate groups: First received AGE (100 mg/kg, IP), second group as the control group received phosphate buffered saline. Treatments were carried out 3 times/week. Tumor growth was measured and morbidity was recorded. Subpopulations of CD4+/CD8+ T cells were determined using flow cytometry. WEHI-164 cell specific cytotoxicity of splenocytes and in vitro production of interferon gamma (IFN-γ) and interleukin-4 cytokines were measured. The mice received AGE had significantly longer survival time compared with the control mice. The inhibitory effect on tumor growth was seen in AGE treated mice. The CD4+/CD8+ ratio and in vitro IFN-γ production of splenocytes were significantly increased in AGE group. WEHI-164 specific cytotoxicity of splenocytes from AGE mice was also significantly increased at 25:1 E: T ratio. Administration of AGE resulted in improved immune responses against experimentally implanted fibrosarcoma tumors in BALB/c mice. AGE showed significant effects on inhibition of tumor growth and longevity of survival times.

Schedule‐dependent therapeutic efficacy of L19mTNF‐α and melphalan combined with gemcitabine

L19-tumor necrosis factor alpha (L19mTNF-α; L), a fusion protein consisting of mouse TNFα and the human antibody fragment L19 directed to the extra domain-B (ED-B) of fibronectin, is able to selectively target tumor vasculature and to exert a long-lasting therapeutic activity in combination with melphalan (M) in syngeneic mouse tumor models. We have studied the antitumor activity of single L19mTNF-α treatment in combination with melphalan and gemcitabine (G) using different administration protocols in two histologically different murine tumor models: WEHI-164 fibrosarcoma and K7M2 osteosarcoma. All responding mice showed significant reduction in myeloid-derived suppressor cells (MDSCs) and an increase in CD4+ and CD8+ T cells in the tumor infiltrates, as well as significant reduction in regulatory T cells (Treg) at the level of draining lymph nodes. What is important is that all cured mice rejected tumor challenge up to 1 year after therapy. Targeted delivery of L19mTNF-α synergistically increases the antitumor activity of melphalan and gemcitabine, but optimal administration schedules are required. This study provides information for designing clinical studies using L19mTNF-α in combination with chemotherapeutic drugs.Targeted delivery of L19mTNF-α synergistically increases the antitumor activity of melphalan and gemcitabine, but optimal administration schedule requires a pretreatment with L19mTNF-α otherwise an antagonistic effect could occur. This study provides information for designing clinical studies using L19mTNF-α in combination with chemotherapeutic drugs.

Immunomodulatory activity of aged garlic extract against implanted fibrosarcoma tumor in mice

Background:Garlic is known as a medicinal herb with broad therapeutic properties ranging from antibacterial to anticancer and even anticoagulant.Aim:Current study was designed to evaluate antitumor effects of aged garlic extract (AGE) on fibrosarcoma tumor in BALB/c mice.Materials and Methods:WEHI-164 fibrosarcoma cells were implanted subcutaneously on day zero into right flank of 40 BALB/c mice aged eight weeks. Mice were randomly categorized in two separate groups: 1st received AGE (100 mg/kg, intraperitoneally), 2nd group as control received phosphate buffered saline, (PBS). Treatments were done three times per week. Tumor growth was measured and morbidity was recorded. Subpopulations of CD4+/CD8+ T cells were determined using flow cytometry. WEHI-164 cell specific cytotoxicity of splenocytes and in vitro production of gamma-interferon, (IFN-γ) and Interleukin-4, (IL-4) cytokines were measured.Results:The mice received AGE had significantly longer survival time compared to control mice. The inhibitory effect on tumor growth was seen in AGE treated mice. The CD4+/CD8+ ratio and in vitro IFN-γ production of splenocytes were significantly increased in AGE group.Conclusions:Administration of AGE resulted in improved immune responses against experimentally implanted fibrosarcoma tumors in BALB/c mice. AGE showed significant effects on inhibition of tumor growth and longevity of survival times.

English

Tumor invasion and metastasis are the main causes of cancer death. Matrix metalloproteinases (MMPs) are endopeptidases which degredate the extracellular matrix proteins. MMPs have an important role in tumor progression and metastasis.&nbsp;Fibrosarcoma has been recognized as a highly metastatic cancer which its current therapeutic methods have not been very successful. Medicinal plants are widely used in treatment of cancers. Spearmint is a herb with well known anti-tumor activities. In the present study, the effect of hydro extract of spearmint on MMP-2/9 activities in Wehi-164 fibrosarcoma cells has been evaluated&nbsp;in vitro. Wehi-164 cells were cultured&nbsp;in Roswell Park Memorial Institute (RPMI) medium with 10% fetal bovine serum (FBS). Then the cells at logarithmic growth phase were incubated with different concentrations of hydro extract of spearmint (0.1 to 10 mg/ml) in the presence or absence of&nbsp;phorbol myristate acetate (PMA) (25 ng/ml) for 24, 48 and 72 h. The MMP-2/MMP-9 activity in conditioned medium was evaluated by gelatin zymography.Spearmint aqueous extract significantly reduced the MMP-2 and PMA-induced MMP-9 activity in fibrosarcoma Wehi-164 cells dose dependently. The aqueous extract of spearmint showed inhibitory effect on MMP-2/MMP-9 activity in Wehi-164 fibrosarcoma cells. So, the anti-tumor effects of spearmint may be in part due to its inhibitory effects on MMP-2/MMP-9 activity. Evaluation of effective component (s) in the spearmint extract with anti-MMP activity and their mechanism of action could be useful in designing novel natural MMP inhibitors. &nbsp; Key words:&nbsp;Spearmint, matrix metalloproteinases, fibrosarcoma.

&amp;lt;i&amp;gt;Toxoplasma gondii&amp;lt;/i&amp;gt; But Not &amp;lt;i&amp;gt;Leishmania major&amp;lt;/i&amp;gt; or &amp;lt;i&amp;gt;Trichomonas vaginalis&amp;lt;/i&amp;gt; Decreases Cell Proliferation and Increases Cell Death on Fibrosarcoma Cancer Cells in Culture Medium

It has been shown that some parasites interfere with malignant tumor cells growth in vivo or in vitro. In this work anticancer activity of three live protozoan parasites; Toxoplasma gondii, Trichomonas vaginalis and Leishmania major on fibrosarcoma cells growth has been investigated in cell culture medium. In this experimental study, WEHI-164 fibrosarcoma cells treated with alive Toxoplasma gondii tachyzoite, Trichomonas vaginalis trophozoite or Leishmania major promastigote as case groups or left intact as control groups. Following 24 hours incubation the number of cells, lactate dehydrogenase (LDH) and apoptosis were determined in case and control groups. Toxoplasma gondii tachyzoite decreased cell proliferation and increased cell lyses’ but it did not induce apoptosis. Trichomonas vaginalis or Leishmania major didn’t show any effects on cell proliferation, cell lyses or apoptosis. Therefore Toxoplasma gondii may have anticancer activity and further works is recommended to understand the mechanisms of action.

Hydatid Cyst Protoscolices Induce Cell Death in WEHI-164 Fibrosarcoma Cells and Inhibit the Proliferation of Baby Hamster Kidney FibroblastsIn Vitro

Both in vitro and in vivo models have demonstrated that some parasites can interfere with tumor cell growth. The present study investigates the anticancer activity of hydatid cyst protoscolices on WEHI-164 fibrosarcoma cells and baby hamster kidney (BHK) fibroblast cells in vitro. Those above two cell types were treated with live hydatid cyst protoscolices or left untreated for control groups. After 48 h, lactate dehydrogenase (LDH) and cell counts were assayed for both treated cells and control groups. Following treatment with hydatid cyst protoscolices, cell proliferation of both cell types was inhibited, and lysis of fibrosarcoma cells increased. Based on these results, it appears that hydatid cyst protoscolices have strong anticancer activity, and additional studies are needed to further clarify the mechanisms of this activity.

Cytotoxic effect of Mentha spicata aqueous extract on cancerous cell lines in vitro

Mentha spicata is a herb with several biological properties. Cytotoxicity of essential oils of M. spicata on some cancer cells has been reported. In this study the cytotoxicity of aqueous extract of M. spicata on two tumor cell lines (Wehi-164 fibrosarcoma and U937 leukemic monocyte) has been evaluated in vitro . Wehi-164 and U937 cells were separately cultured in RPMI with 10% FBS. Then the cells at logarithmic growth phase were incubated in the presence of different concentrations of aqueous extract of M. spicata (0.1 to 10 mg/ml) at 24, 48 and 72 h periods. The cell proliferation was assessed with trypan blue dye exclusion and MTT assays. Aqueous extract of M. spicata significantly reduced the proliferation of Wehi-164 and U937 cells dose and time- dependently. The LD 50 values of M. spicata extract were 5.97, 4.63 and 4.77 mg/ml for the Wehi-164 cells and 5.6, 5.3 and 4.84 mg/ml for the U937 cells, after 24, 48 and 72 h treatment respectively. Aqueous extract of M. spicata showed cytotoxic effect in mouse fibrosarcoma Wehi-164 and human monocytic U937 cells. Thus, M. spicata could have potential anti-tumor activity. In vivo studies as well as identification of effective components of M. spicata with anti-cancer activity and their exact mechanism of action could be useful in designing new anti-cancer therapeutic agents.

A Comparative Study upon the Therapeutic Indices of Some Natural and Synthetic Anti-inflammatory Agents.

THE AIM OF THE PRESENT STUDY WAS TO COMPARE THE THERAPEUTIC INDICES OF SEVERAL AGENTS USED IN TREATMENT OF INFLAMMATORY CONDITIONS WHICH INCLUDED: Vitamin E, hydro-alcoholic extracts of Glycyrrhiza glabra, Matricaria aurea, dexamethasone, piroxicam and diclofenac using Wehi-164 fibrosarcoma cells. Cytotoxicity evaluation was based on vital dye exclusion assay. Matrix-metalloproteinases inhibition (MMPI) was assessed by gelatinase zymography method. The collected data were used to estimate the IC50 (50% MMPI concentration), LC50 (50% cytotoxicity concentration) and the therapeutic index (LC50/IC50). Among the natural anti-inflammatory agents used, M. aurea was the least toxic and the most effective inhibitor of MMP. Vitamin E not only increased MMP activity, but also was the most toxic of all the agents tested. Next in terms of toxicity to vitamin E was G. glabra. Diclofenac was more toxic than both piroxicam and dexamethasone. Findings from this study suggest that medicinal plants reputed to have anti-inflammatory properties are not equally effective and safe. In order to assess the implications of these findings, further in vitro and in vivo studies are needed.

Natural Cytotoxicity of NC‐2+ Cells Against the Growth and Metastasis of WEHI‐164 Fibrosarcoma

We have previously reported a new receptor (NC-2) for natural cytotoxicity (NC) on murine leucocytes, identified by monoclonal antibody D9 (mAb D9). Pretreatment of mouse spleen cells with different concentrations of mAb D9 in vitro blocked NC against WEHI-164, whereas natural killing (NK) activity against YAC-1 was unaffected. This paper reports the immune surveillance against the growth of WEHI-164 tumour cells in mice by NC-2(+) Cells. The kinetics of in vivo reduction in NC activity were investigated by treating BALB/c and (CBA × C57BL/6) F1 mice with a single injection of 40 μg of mAb D9 and monitoring splenic NC activity by (51) Cr-release assay at intervals from 24 h to 3 weeks. Control mice were injected with OKT8 irrelevant antibody. Results showed a significant (P < 0.05) reduction in splenic NC activity within 24 h which persisted for up to 1 week. Similar results were also obtained when (CBA × C57BL/6) F1 mice were employed (P<0.001). In vivo tumour studies were undertaken to investigate the role of NC-2(+) cells in surveillance against tumour growth and metastasis of the WEHI-164 fibrosarcoma. When syngeneic BALB/c mice were injected with 40 μg of mAb D9 and then challenged with 5 × 10(5) WEHI-164 cells, results showed significantly increased growth rate of the transplanted WEHI-164 fibrosarcoma and tumour nodules in the lungs of animals, when compared to control mice with normal NC activity. Our data support an innate surveillance in metastasis and growth of WEHI-164 fibrosarcoma in mice.