Generation of T helper cells in vitro. IV. F1 T helper cells primed with antigen-pulsed parental macrophages are genetically restricted in their antigen-specific helper activity.

A sequential culture technique for the in vitro induction and subsequent assay of T helper cells is employed to examine the histocompatibility requirements for antigen recognition by murine T helper cells. F1 T cells are primed in vitro with antigen-pulsed parental strain macrophages and tested for antigen-specific helper activity in cultures containing anti-Thy 1.2 serum and C treated spleen cells from hapten-primed parental or F1 mice. A semiallogenieic system is used and appropriate controls are included to avoid possible complicating effects of allogeneic interactions. The results indicate that F1 T helper cells preferentially stimulate carrier-specific anti-hapten plaque-forming cell responses in spleen cells which are H-2 identical with the macrophage used initially to prime the T cells. Parental spleen cell cultures do not respond to F1 T helper cells which were primed with the other parental strain macrophage. Supplementing this culture with macrophages which are histocompatible with those used to prime the F1T cells is sufficient to restore T helper cell activity. Thus, the genetic restriction described here is between the primed T cell and the macrophage used to elicit secondary responses and not between the T cell and B cell. The results in this semiallogeneic system, however, do not rule out the possibility of additional allogeneic genetic restrictions in the subsequent interaction of T cells with B cells.     

IFN-gamma-dependent recruitment of mature CD27(high) NK cells to lymph nodes primed by dendritic cells

NK cells have been proposed to be an initial source of IFN-gamma that supports either Th1 or CTL priming. Although NK cells reside in naive lymph nodes (LN) at a very low frequency, they can be recruited into LN draining sites of infection, inflammation, or immunization where they potentially influence adaptive immunity. In this study, we report that mature CD27(high) NK cells are predominantly recruited into the draining LN following dendritic cell (DC) challenge. Importantly, the recruitment of the CD27(high) NK cell subset in the draining LN was dependent on host IFN-gamma and the activation status of NK cells. Endogenous epidermal DC migration induced by hapten challenge also triggers NK cell recruitment to the draining LN in an IFN-gamma-dependent mechanism. Thus, our results identify that CD27(high) NK cells are the dominant population recruited to the draining LN and NK cell recruitment requires endogenous IFN-gamma in coordinating with DC migration.     

Lymphocyte specificity to protein antigens. I. Characterization of the antigen-induced in vitro T cell-dependent proliferative response with lymph node cells from primed mice.

An in vitro assay which measures antigen-induced proliferation of primed murine lymph node cells is described. The response is mediated by T eclls since it can be obtained by using nylon wool-passed lymphocytes (less than 1% Ig+ cells) and it can be abolished by treatment with anti-Thy 1.2 and C. Furthermore, LN cells from nu/nu mice injected with antigen do not demonstrate antigen-induced proliferation in contrast to the response observed in euthymic littermate controls. Other relevant parameters of this proliferative assay include the observations that the response is highly antigen specific, can be seen as early as 4 days and as late as 60 days after in vivo priming, is restricted to the use of certain sets of LN when animals are injected subcutaneously at the base of the tail, and can be seen with LN cells from mice primed with antigen in either CFA or ICFA. The ease of the assay coupled with its specificity and quantitative dimensions provides a direct and simple method to evaluate processes involved in antigen-induced murine T lymphocyte activation.     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

Modulation of F1 cytotoxic potentials by GvHR. Host- and donor-derived cytotoxic lymphocytes arise in the unirradiated F1 host spleens under the condition of GvHR-associated immunosuppression

As an approach to dissect complex cellular events that lead to GvHR-associated immune disorders, we followed cytotoxic activities, including NK cytotoxicity, in the spleens of unirradiated F1 hosts undergoing GvHR induced by parental spleen cells. Spleen cells of (B10 X DBA/2)F1 or (B10 X AKR/J)F1 hosts undergoing GvHR induced by parental B10 spleen cells displayed a prompt and marked increase in NK cell activity within 36 hr, and the heightened activity lasted until day 8. The activity then declined abruptly and disappeared on day 12 of GvHR. Inversely, donor B10-derived CTL specifically directed to the opposite parental alloantigens of the F1 hosts emerged in these F1 host spleens on day 8, and the CTL activity reached a peak on day 12 when the host NK cell activity disappeared. During the period that the donor-derived anti-host CTL were present, these F1 host spleen cells lost not only NK cell activity but also the ability to mount in vitro CTL responses. In contrast, the respective F1 strain mice undergoing GvHR induced by the parental DBA/2 or AKR/J spleen cells showed only transient but marked increases in NK cell activity during the initial 36 hr, and then the activity decreased gradually to return to the normal level on day 10. In such GvHR F1 host spleens, donor-derived CTL could never be detected, and the spleen cells showed normal in vitro CTL responsiveness during the entire observation period of 16 days. These results are discussed from the viewpoint of genetically defined cellular events that lead to the GvHR-associated immune disorders.     

Augmentation of Mouse Natural Killer Cell Activity by Lactobacillus casei and Its Surface Antigens

Lactobacillus casei YIT 9018 (LC 9018) augmented the natural killer (NK) cell activity of spleen cells from inbred BALB/c mice injected intravenously with LC 9018 or intraperitoneally with polyinosinate-polycytidylate. Augmentation of this activity by LC 9018 was also observed in male C3H/He, CBA/N, and C57BL/6 mice. The spleen cells exhibited no cytolytic activity against P815, a cell line insensitive to NK cells. The cytolytic activity of the spleen cells increased 2 days after the injection of 250 μg of LC 9018/mouse, peaked on day 3, and gradually declined thereafter. The increase caused by LC 9018 was also observed in normal and Meth A-bearing mice. In vitro treatment with anti-asialo GM1 antibody plus complement completely-abrogated the LC 9018-augmented murine NK cell activity. The NK activity on the 3rd day after LC 9018 injection was reduced by in vitro treatment with anti-Thy 1.2 monoclonal antibody plus complement to half of that observed when treatment was with complement alone. This suggests that there were two populations of NK cells in the spleen cell suspension derived from LC 9018-treated mice. One population was asialo GM1-positive and Thy 1-negative, the other was asialo GM1-positive and Thy 1-positive.     

Characterization of subpopulations of T lymphocytes: I. Separation and functional studies of peripheral T-cells binding different amounts of fluorescent anti-Thy 1.2 (theta) antibody using a fluorescence-activated cell sorter (FACS)

Peripheral T lymphocytes in mice can be distinguished by the presence of the Thy 1.2 (theta) cell surface antigen. The fluorescence-activated cell sorter (FACS) was used to analyze and separate T-cells from peripheral lymphoid cell suspensions after incubation with fluorescein-labeled anti-Thy 1.2 (F anti-Thy 1.2). Stained cells were markedly reduced in nu/nu mice, in mice carrying the Thy 1.1 allele (theta-AKR), and were not seen after incubation with anti-Thy 1.2 that had been absorbed with CBA brain. According to these criteria, the stained cells were termed “T lymphocytes.”Among the T lymphocytes, there was considerable heterogeneity of fluorescent staining. The FACS was used to separate T-cells from other cells and further to separate T-cells with high intensity F anti-Thy 1.2 fluorescence (bright T-cells) from those with less F anti-Thy 1.2 fluorescence (dull T-cells). Separated bright T and dull T lymphocytes were shown to have several different functional properties. Dull T-cells appeared more sensitive to small doses of ALS in vivo, homed to lymph node in higher proportions than did bright T-cells, and were not affected by the short-term effects of thymectomy in adult life. Bright T lymphocytes, by contrast, were resistant to the in vivo effects of ALS, homed preferentially to spleen rather than lymph node in irradiated hosts, and were reduced shortly after adult thymectomy. Separated populations of bright and dull T-cells showed reduced ability to produce cytotoxic activity after in vitro sensitization, while mixtures of these two subpopulations of T-cells produced synergistic cytototoxic responses. The ontogenic and functional implications of these findings are discussed.     

Suppressor T cells in thymus-reconstituted nude mice: regulation of mitogen-induced transformation

A population of glass-wool-adherent splenic cells which could suppress the response of other spleen cell populations to T-cell mitogens was isolated from thymus-reconstituted nude mice. Such adherent cells were characterized as sensitive to anti-Thy 1.2 and complement treatment. Glass-wool-adherent cells from athymic mice do not have suppressor activity to self or normal littermate NAC; however, these mice possess precursor suppressor cells as demonstrated by isolation of glass-wool-adherent T regulatory cells in thymus-grafted nude mice. Such cells are generated in either freshly obtained or in vitro cultured thymus. Evidence for suppressor T cells of host genotype was supported by their sensitivity to host-specific anti-Thy serum treatment as well as their generation in alymphoid thymus grafts. Prior anti-Thy 1.2 treatment of GAC partially removed the suppressor activity: however, macrophages and B lymphocytes were shown not to be secondary regulatory cells or suppressor mediators, thus mature T lymphocytes with low amounts of Thy 1.2 antigen may be responsible for this residual suppression. Further characterization of GAC indicates that active cell growth is required for their regulatory function, as irradiation removed the suppressor activity. This report provides evidence for the presence of a T-lymphocyte subpopulation which has a regulatory function and requires a thymus in the generation of these cells.     

Immune suppression in vivo with antigen-modified syngeneic cells. II. T cell-mediated nonresponsiveness to fowl gamma-globulin.

Intravenous administration of syngeneic spleen cells coupled with the palmitoyl derivative of fowl gammma-globulin (p-F gamma G) results in a profound state of F gamma G-specific tolerance in C57BL/6 mice. Administration of p-F gamma G coupled syngeneic cells specifically reduces both the primary and secondary hapten and carrier-specific PFC responses to TNP-F gamma G. Since the haptenic response is affected, the tolerance functions at the level of the F gamma G-specific helper T cell. As few as 10(3) p-F gamma G spleen cells carrying only 1 ng of p-F gamma G can induce tolerance. At least a 2-day-induction period is required. This nonresponsiveness is long lived, lasting over 120 days. Spleen cells from tolerized mice can transfer suppression to normal syngeneic recipients. Treatment of tolerant spleens with anti-Thy 1.2 antiserum + C eliminates the suppressor cell activity. In addition, thymocytes and purified splenic T cells from tolerized mice can transfer suppression to normal recipients. Thus, at least a component of this nonresponsiveness is mediated by suppressor T cells. The requirement of antigen association with cell membrane components and the general applicability of this method of inducing T cell nonresponsiveness are discussed.     

Adherent cell function in murine T lymphocyte antigen recognition. I. A. macrophage-dependent T cell proliferation assay in the mouse.

The data in this report describe a T cell proliferation assay with nylon wool column-purified murine lymph node lymphocyte from animals immunized by footpad injection of antigen in CFA. It was found that the in vitro immune response of sensitized T cells to soluble protein antigens was functionally dependent on the presence of adherent cells, more specifically macrophages, at all concentrations of in vitro antigen challenge. The response was due to T cells in that cytotoxic treatment of the immune lymphocyte cells with anti-Thy 1.2 serum and complement effectively eliminated the antigen-specific DNA synthetic responses. The antigen-specific proliferation of murine lymphocytes depleted of adhereent cells could not be reconstituted with either guinea pig macrophages nor murine fibroblasts, indicating the existence of species and cell type specificity. In contrast to previous observations in the guinea pig, soluble products of cultured adherent cells could at least partially replace the function of intact macrophages in the response to antigen.     

Low density of Thy 1 antigen on mouse effector cells mediating natural cytotoxicity against tumor cells.

Mouse effector cells mediating natural cell-mediated cytotoxicity against tumor cells were found to contain a low density of Thy 1 antigen. Treatment of nude spleen cells, or spleen cells from mice in which natural reactivity was boosted, with anti-Thy 1.2 plus complement resulted in a substantial decrease in cytotoxicity. The spontaneous cytotoxic reactivity of young, thymus-bearing mice was resistant to such treatment, but repeated exposure to anti-Thy 1.2 plus complement did cause a decrease in lytic activity. By use of congenic anti-Thy 1.2 and effector cells from mice congenic for Thy 1, the effects of the treatment were shown to be specific for Thy 1.2 antigen.     

Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function

alpha-Dystroglycan (alpha-DG) is a laminin-binding protein and member of a glycoprotein complex associated with dystrophin that has been implicated in the etiology of several muscular dystrophies. To study the function of DG, C2 myoblasts were transfected stably with an antisense DG expression construct. Myotubes from two resulting clones (11F and 11E) had at least a 40-50% and 80-90% reduction, respectively, in alpha-DG but normal or near normal levels of alpha-sarcoglycan, integrin beta1 subunit, acetylcholine receptors (AChRs), and muscle-specific kinase (MuSK) when compared with parental C2 cells or three clones (11A, 9B, and 10C) which went through the same transfection and selection procedures but expressed normal levels of alpha-DG. Antisense DG-expressing myoblasts proliferate at the same rate as parental C2 cells and differentiate into myotubes, however, a gradual loss of cells was observed in these cultures. This loss correlates with increased apoptosis as indicated by greater numbers of nuclei with condensed chromatin and more nuclei labeled by the TUNEL method. Moreover, there was no sign of increased membrane permeability to Trypan blue as would be expected with necrosis. Unlike parental C2 myotubes, 11F and 11E myotubes had very little laminin (LN) on their surfaces; LN instead tended to accumulate on the substratum between myotubes. Exogenous LN bound to C2 myotubes and was redistributed into plaques along with alpha-DG on their surfaces but far fewer LN/alpha-DG plaques were seen after LN addition to 11F or 11E myotubes. These results suggest that alpha-DG is a functional LN receptor in situ which is required for deposition of LN on the cell and, further, implicate alpha-DG in the maintenance of myotube viability.     

Immune response to the p-azobenzenearsonate (ABA)-GAT conjugate. II. Hapten-specific T cells induced with ABA-GAT in GAT responder X nonresponder F1 hybrids are restricted to the nonresponder haplotype

Immunization of mice with the ABA-GAT conjugate stimulates GAT-specific T helper cells in GAT-responder animals and ABA-specific helpers in nonresponders. Unexpectedly, immunization of (responder X nonresponder) F1 mice, which have the GAT-responder phenotype, leads to the recruitment of both ABA- and GAT-specific clones of T helper lymphocytes. The GAT-reactive population is restricted to the haplotype of the responder parent (Iak), whereas ABA-specific T cells are mostly restricted to the nonresponder one (Ias). This is demonstrated by the ability of monoclonal antibodies to parental la antigens to inhibit T cell proliferation to GAT or ABA-Tyr in vitro. Consistently, ABA-GAT-primed F1 T cells can only activate nonresponder B cells to proliferate in the presence of ABA-Tyr and responder B lymphocytes in the presence of GAT. Furthermore, F1 T cells seem to recognize both ABA and GAT epitopes only in association with molecules encoded by the I-A subregion. Analysis of ABA-specific F1 T cell lines generated by in vitro stimulation with ABA-Tyr or ABA-GAT demonstrates a competition between GAT- and ABA-specific T cells present in the hybrid T cell repertoire and restricted to the same parental I-Ak molecule. The results indicate that F1 macrophages can present both ABA and GAT epitopes to T cells in association with the two parental and hybrid Ia determinants. It seems unlikely that the absence of GAT-specific T cells restricted to the nonresponder I-A in the F1 is due to suppressor T cells. Thus, the competition model that we propose, to explain the selective F1 T cell response to ABA-GAT, leads us to believe that GAT nonresponder animals may lack clones capable of recognizing, with a high affinity, I-As + GAT.     

Specific inhibition of hybrid resistance in F1 hybrid mice pretreated with parent-strain spleen cells. II. Evidence for an Hh-1 antigen-specific tolerization

Lethally irradiated F1 mice reject bone marrow graft from H-2b parents. In a previous paper we showed that pretreatment of F1 hybrid with H-2b parental spleen cells abrogates this hybrid resistance (HR) to parental bone marrow growth by inducing a Thy-1+Lyt-1+2- nylon-adherent suppressor cell. We studied the mechanism of induction of this suppressor cell. Two hypotheses were tested; both were based on the observation that parental spleen cells when injected into a F1 hybrid, recognize the alloantigens of the opposite parent and proliferate; the proliferation of these Hh-1+ cells may result in an overload of the pretreated F1 hybrids with Hh-1 Ag, and in the development of a graft-vs-host reaction that is followed by a non-specific immunodeficiency (GVHID). Thus abrogation of HR could be due to either a tolerization with high doses of Hh-1 Ag or the GVHID. Our results show that abrogation of HR does not correlate with the GVHID because 1) it is induced after pre-treatment with H-2b parental cells only, whereas GVHID is observed after injection with cells from either of the two parents; and 2) it is induced in several conditions where GVHID does not occur; after pre-treatment with 1000-rad-irradiated or T-cell depleted or only class I incompatible spleen cells or with spleen cells from nude parents as well as after pre-treatment with H-2b bone marrow cells. HR is overcome by the injection of H-2Db homozygous or of cross-reactive H-2Ds homozygous cells only. However, although pretreatment with H-2Db homozygous spleen cells is necessary, it is not sufficient for an efficient overcoming of HR. Indeed enhancement of H-2b bone marrow growth after pre-treatment with 1000-rad-irradiated, T-cell depleted or nude parent spleen cells is very short-lasting and never reaches the level observed after pre-treatment with normal spleen cells. We conclude that inhibition of HR in F1 hybrids pretreated with parental spleen cells is not a consequence of a GVHID but of a specific tolerization with Hh-1 Ag; however, the HR is inhibited more consistently when inoculum used for the pretreatment contains fully immunocompetent T cells. The role of the immunocompetent parental T cells in abrogation of HR is discussed.     

Interactions between CD3 and Thy1 T cell activation pathways: blockade of CD3-mediated T lymphocyte activation induced by immobilized anti-Thy1 antibodies.

Resting murine T cell activation induced by either CD3 complexes or Thy1 molecules was investigated in vitro, using surface-bound anti-CD3 mAb as the stimulus. One mitogenic anti-Thy 1 mAb (G7) lost mitogenicity when presented to T cells immobilized on a plastic surface, even in the presence of phorbol ester. Moreover, T cell activation induced by immobilized anti-CD3 was potently blocked by coimmobilized anti-Thy 1 mAb. Nonmitogenic anti-Thy 1 mAb also blocked CD3-induced activation when coimmobilized with anti-CD3. Control experiments showed that anti-Thy 1 specifically blocked T cell activation, even in the presence of measurable and functional concentrations of plastic-bound anti-CD3. Coimmobilized anti-Thy 1 potently blocked IL2 secretion stimulated by anti-CD3. Addition of exogenous rIL2 completely prevented anti-Thy 1-mediated blockade. On the other hand, while completely blocking T cell proliferation, immobilized anti-Thy 1 only partially blocked secretion of IL3-like activity by the T cells. One IgM anti-Thy 1 mAb (2A3) induced secretion of IL3-like activity by T cells when immobilized in the absence of bound anti-CD3. These results indicate that extensive aggregation of Thy 1 molecules delivers a potent negative signal which antagonizes CD3-mediated T cell activation and growth.     

Expression of Thy 1 thymocyte differentiation antigen in mouse mammary cancer cells in vitro

Allogeneic AKR-anti C3H Thy 1.2 antigen serum and monoclonal anti-Thy 1.2 and anti-Thy 1.1 antigen antibodies were used to study the expression of lymphocyte differentiation antigen in a clonal mammary carcinoma cell line originated from a GR/mt stable cell line. Both allogeneic antiserum and monoclonal anti-Thy 1.2 (but not Thy 1.1) antibody were active with the hormone-treated fixed cells in an indirect immunofluorescence test. However, antigen on the cellular membranes could be detected only with the use of allogeneic (but not with monoclonal antibody) anti-Thy 1.2 serum.     

Immunosuppression of normal lymphoid cells by serum from mice undergoing chronic graft-vs-host disease.

Spleen cells from F1 mice undergoing chronic graft-vs-host (GVH) reaction, induced by injection of parental cells, were shown to be immunosuppressed since their in vitro responses to the mitogens concanavalin A (Con A) and bacterial lipopolysaccharide (LPS) were substantially lower than control animals. Serum, from mice undergoing GVH, when cultured in vitro with normal spleen cells was immunosuppressive. The proliferation response to Con A and allogeneic cells of normal syngeneic, allogeneic, and parental spleen cells was 90% suppressed when serum from mice undergoing chronic GVH was added in comparison to the addition of serum from untreated F1 mice. Similarly, the in vitro antibody response to a T-dependent antigen was impaired; however, the antibody response to a T-independent antigen was not impaired. These results indicate that T cell functions are more sensitive than are B cell functions to immunosuppressive factors in the serum of mice undergoing GVH.     

Progression from acute to chronic disease in a murine parent-into-F1 model of graft-versus-host disease

The parent-into-immunocompetent-F(1) model of graft-vs-host disease (GVHD) induces immune dysregulation, resulting in acute or chronic GVHD. The disease outcome is thought to be determined by the number of parental anti-F(1) CTL precursor cells present in the inoculum. Injection of C57BL/6 (B6) splenocytes into (B6 x DBA/2)F(1) (B6D2F(1)) mice (acute model) leads to extensive parental cell engraftment and early death, whereas injection of DBA/2 cells (chronic model) results in little parental cell engraftment and a lupus-like disease. This study demonstrated that injection of BALB/c splenocytes into (BALB/c x B6)F(1) (CB6F(1)) mice resulted in little engraftment of parental lymphocytes and the development of lupus as expected. Injection of B6 splenocytes into CB6F(1) initiated an initial burst of parental cell engraftment similar to that of B6 into B6D2F(1). However, the acute disease resolved, and the CB6F(1) mice went on to develop chronic GVHD with detectable Abs to ssDNA, dsDNA, and extractable nuclear Ags. Limiting dilution CTL assays determined that B6 splenocytes have CTL precursor frequencies of 1/1000 against both CB6F(1) and B6D2F(1), whereas DBA/2 and BALB/c splenocytes have a CTL precursor frequency of 1/20,000 for their respective F(1)s. The Th cell precursor frequency for B6 anti-DBA/2 was 3-fold higher than that for B6 anti-BALB/c determined by limiting dilution proliferation assays. These results indicate the importance of adequate allospecific helper as well as effector T cells for the induction and maintenance of acute GVHD in this model, and presents an unexpected model in which initial acute GVHD is replaced by the chronic form of disease.     

Alteronol inhibits the invasion and metastasis of B16F10 and B16F1 melanoma cells in vitro and in vivo

Aims

The purpose of this study is to evaluate the anti-metastatic effects of alteronol on melanoma B16F10 and B16F1 cells in vitro and in vivo.

Main methods

Melanoma B16F1 and B16F10 cells were cultured in vitro. Cell proliferation was analyzed via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell migration and invasion were evaluated via wound healing and transwell chamber assays. The activity of matrix metalloproteinase 2 (MMP-2) in culture supernatants was assessed via gelatin zymography. The expression of MMP-2 and TIMP-2 were detected via enzyme-linked immunosorbent assay (ELISA) assay. The anti-metastatic ability in vivo was detected through experimental lung metastasis.

Key findings

The data indicate that alteronol can inhibit the proliferation, invasion, and migration of B16F1 and B16F10 cells in vitro and in vivo, decrease the activity and expression of MMP-2, enhance the expression level of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), and inhibit the experimental lung metastasis of B16F1 and B16F10 cells.

Significance

Although alteronol and taxol are obtained from the same source, these substances do not destroy the rare resource; the mechanisms of them on tumor growth inhibition are different. Conversely, alteronol treatment had lesser effects on normal cells revealing for a selective property and a strong competitive advantage.