K1735-M2 Melanoma Research Articles

Abstract IA30: Intrinsic and extrinsic contributions of mitochondrial DNA to metastatic efficiency: A genetic explanation for disparities in metastasis efficiency?

Abstract Single-nucleotide polymorphisms (SNP) in nuclear and mitochondrial DNA are used to define clades (or races) in people, as well as different strains in mice (PMID: 27383787). Previous studies showed that nuclear SNP determine metastasis efficiency; we hypothesized that mtDNA SNP could also play roles in tumorigenicity and metastasis. Mitochondrial Nuclear Exchange (MNX) mice, created by transferring an oocyte nucleus from strainx into an enucleated oocyte from strainy (PMID: 27840835), show that mammary tumor formation and metastasis are regulated by inherited mitochondrial polymorphisms when MNX mice were bred to MMTV-PyMT or MMTV-HER2 mice (PMID: 26471915; 29070615). Since stromal compartments also possess changed mitochondria in MNX mice, we hypothesized that mitochondrial SNP in noncancer compartments could exert effects on tumor formation or metastasis in addition to genetic (cell autonomous) changes. Syngeneic tumor cells were injected into MNX mice with the same nuclear (and, therefore, same histocompatibility) background. Experimental metastasis was compared between wild-type and MNX mice (i.e., with same or different mtDNA backgrounds, respectively). E0771 mammary carcinoma and B16-F10 melanoma cells (both syngeneic to C57BL/6J), formed significantly (P<0.01) more lung metastases in C57BL/6J-mtMNX(C3H/HeN). K1735-M2 melanoma cells (syngeneic to C3H/HeN) formed significantly (P<0.05) fewer lung metastases in C3H/HeNmtMNX(C57BL/6J) mice. These results have been replicated ≥3 times using >10 mice per experiment. C57BL/6J mitochondria confer resistance to metastasis in both cell autonomous and non-cell autonomous experiments. Basal metabolic and ROS differences comparing mouse embryonic fibroblasts isolated from wild-type and MNX mice exist and may be responsible for the stromal effects. Likewise, significant and statistically significant differences in MNX mice for nuclear DNA methylation (PMID: 28663334) and epigenetic marks (J. McGuire & D.R. Welch, in preparation), immune (T.C. Beadnell & D.R. Welch, in preparation) and microbota (S.J. Manley & D.R. Welch, in preparation) profiles are observed. Together, our findings highlight the striking influences that mitochondrial haplotypes can exert on tumorigenicity and metastasis via both intrinsic and extrinsic mechanisms. These findings also suggest that mitochondrial SNP could serve, in part, as a genetic basis to explain racial disparities with regard to cancer aggressiveness. Support: Susan G. Komen for the Cure (SAC11037), Natl. Fndn. Cancer Res., CA168524, W81XWH-18-1-0450, GM103418. Citation Format: Danny R. Welch. Intrinsic and extrinsic contributions of mitochondrial DNA to metastatic efficiency: A genetic explanation for disparities in metastasis efficiency? [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr IA30.

Abstract SY37-03: Mitochondrial genetic contributions to metastatic efficiency

Abstract Complex phenotypes, like tumorigenicity or metastasis, require coordinated expression of multiple genes. Additionally, the contributions of some of those genes can be affected by polymorphisms in the protein coding or regulatory sequences (PMID: 22257951). The pathobiology of metastasis remains poorly understood because both intrinsic (i.e., genetic) and extrinsic (i.e., tumor-microenvironment interactions) are involved. A growing body of data indicates demonstrate that there are underlying genetic components that govern the processes involved in cancer spread and colonization. To assess the contributions of mitochondrial genetics in metastasis, we created Mitochondrial Nuclear Exchange (MNX) mice by transferring an oocyte nucleus from strainx into an enucleated oocyte from strainy, and showed that mammary tumor formation and metastasis are regulated by inherited mitochondrial polymorphisms in an oncogenic driver-dependent manner (PMID: 26471915; PMID: 29070615). Since stromal compartments also possess changed mitochondria in the MNX mice, we hypothesized that mitochondrial polymorphisms in non-cancer cell compartments could exert effects on tumor formation and/or metastasis in addition to genetic (cell autonomous) changes. Syngeneic tumor cells were injected into MNX mice with the same nuclear, and therefore same MHC, background. Experimental metastasis was compared between wild-type and MNX mice (i.e., with same or different mtDNA backgrounds, respectively). E0771 mammary carcinoma and B16-F10 melanoma cells (both syngeneic to C57BL/6J), formed significantly (P<0.01) more lung metastases in C57BL/6J-mtMNX(C3H/HeN). K1735-M2 melanoma cells (syngeneic to C3H/HeN) formed significantly (P<0.05) fewer lung metastases in C3H/HeNmtMNXC57BL/6J. These results have been replicated ≥3 times using >10 mice per experiment. C57BL/6J mitochondria confer resistance to metastasis in both cell autonomous and non-cell autonomous experiments. Basal metabolic and ROS differences comparing mouse embryonic fibroblasts isolated from wild-type and MNX mice are among mechanisms being explored. Differential gene expression occurred in tissues isolated from wild-type and MNX mice (PMID: 28663334). Additionally, altered immune and microbiota profiles have been observed. Together, our findings highlight striking influences that mitochondrial haplotypes can exert on tumorigenicity and metastasis via both intrinsic and extrinsic mechanisms. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res. and CA168524. Citation Format: Danny R. Welch. Mitochondrial genetic contributions to metastatic efficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY37-03.

Abstract 1696: Mitochondrial haplotype alters metastasis in a non-cell autonomous manner

Abstract Mitochondrial Nuclear Exchange (MNX) mice, created by transferring the nucleus from an oocyte from strain x into an enucleated oocyte of strain y, showed that mammary tumor formation and metastasis can be regulated by inherited mitochondrial polymorphisms (PMID26471915). Besides genetic (cell autonomous) changes observed, we asked whether mitochondrial polymorphisms in non-cancer compartments could exert effects on tumor formation or metastasis. Tumor cells were injected into syngeneic wild type mice and tumor growth and metastasis were compared to similarly injected cells into MNX mice sharing the same nuclear but different mtDNA backgrounds. Orthotopic tumor growth rates were equal for all of the cell lines tested. However, the ability to form experimental lung metastases following i.v. injection were dramatically altered. Compared to injections into wild-type, syngeneic mice, E0771 mammary carcinoma and B16-F10 melanoma cells (both syngeneic to C57BL/6J), formed significantly (P<0.01) more lung metastases in C57BL/6J-mtMNX(C3H/HeN) and K1735-M2 melanoma cells (syngeneic to C3H/HeN) formed significantly fewer lung metastases in C3H/HeNmtMNXC57BL/6J. These results have been replicated at least three times using >10 mice per experiment. Interestingly, C57BL/6J mitochondria confer resistance to metastasis in both cell autonomous and non-cell autonomous experiments. Basal metabolic differences comparing mouse embryonic fibroblasts isolated from wild-type and MNX mice are among mechanisms being explored. Together, our findings highlight the striking influences that mitochondrial haplotypes can exert on tumorigenicity and metastasis via both intrinsic and extrinsic mechanisms. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res, Steiner Family Fund for Metastasis Research, Kansas Bioscience Authority, CA134981, P30-CA168524 Citation Format: Amanda E. Brinker, Carolyn J. Vivian, Danny R. Welch. Mitochondrial haplotype alters metastasis in a non-cell autonomous manner. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1696.

Carriers for metal complexes on tumour cells: the effect of cyclodextrins vs CNTs on the model guest phenanthroline-5,6-dione trithiacyclononane ruthenium(II) chloride

The complex [Ru[9]aneS3(pdon)Cl]Cl (pdon = 1,10-phenanthroline-5,6-dione) was readily obtained from the stoichiometric reaction of Ru[9]aneS3(dmso)Cl2 with pdon. Recrystallisation in ethanol using salicylic acid as a co-crystallisation helper afforded single-crystals suitable for the collection of X-ray diffraction data which afforded a reasonable structural description. Two different kinds of molecular carriers were tested as vehicles for this complex: carbon nanotubes (CNTs) and cyclodextrins. CNTs had an insufficient loading rate for the ruthenium complex at CNT concentrations deemed non-cytotoxic on cultured cells. The cyclodextrin (CD) carriers, β-CD and TRIMEB (standing for permethylated β-CD), were able to form two adducts, studied by powder X-ray diffraction, thermogravimetric analysis (TGA), (13)C{(1)H} CP/MAS NMR and FT-IR spectroscopies. The DNA thermal denaturation studies showed that the complex 1 is able to intercalate with DNA. The in vitro cytotoxicity of the free complex [Ru[9]aneS3(pdon)Cl]Cl (1) and of its two CD adducts (2 and 3) was assessed on both rodent and human cell lines. By using the mouse K1735-M2 melanoma cell line and the non-tumour rat H9c2 cardiomyoblasts, the results showed that 1 and 2 significantly inhibited the growth of the tumour cell line while displaying a good safety profile on cardiomyoblasts. Compound 3 at 100 μM inhibited the proliferation of both cell lines, with a higher activity towards the melanoma cell line. The cytotoxicity of the compounds 1-3 was further assessed on human breast cancer cell lines. Against the MDA-MB-231 line, growth inhibition occurred only with 1 and 3 at the incubation time of 96 h, both with approximate inhibition rates of 50 %; against the MCF-7 line, mild cytotoxicity was observed at 48 h of incubation, with IC50 values calculated above 100 μM for 1, 2 and 3.

Decitabine, independent of apoptosis, exerts its cytotoxic effects on cell growth in melanoma cells

Decitabine is a synthesized cytosine analog that is a potent inhibitor of DNA methylation. There have been a few reports on the in vitro anti-melanoma effect of decitabine or its functional mechanisms. We investigated the anti-proliferation effect of decitabine on the cultured murine melanoma cell line K1735M2. MTT assay showed that decitabine had strong inhibition on melanoma K1735M2 in a time- and dose-dependent manner in vitro. Morphological observation showed that decitabine could induce melanoma K1735M2 cells to produce dendrite-like structures with the increase of decitabine concentration and incubation time. Decitabine could effectively induce K1735M2 cells to differentiate in vitro. Additionally, decitabine could induce a dose-dependent G2/M cell cycle arrest in K1735M2 cells. We provided experimental evidences that the anti-proliferation effect of decitabine on murine K1735M2 melanoma cells was associated predominately with G2/M cell cycle arrest and the induction of differentiation rather than apopotosis.

Preventive Effect of Green Tea Catechins on Experimental Tumor Metastasis in Senescence-Accelerated Mice

Successful avoidance of the immune surveillance system is critical for the development of a blood-borne metastasis. Previous findings suggest that experimental tumor metastasis was enhanced in senescence-accelerated mice prone 10 (SAMP10) due to a reduction in immune surveillance potential with age. In the present study, water containing green tea (GT)-catechins was freely given to SAMP10 mice, and the chemopreventive effect of GT-catechin intake on tumor metastasis was examined. Natural killer cell activity, which is an indicator of immune surveillance potential and is reduced in control mice with age, was maintained by GT-catechin intake. The early accumulation of lung-metastatic K1735M2 melanoma cells in lungs after intravenous injection of the cells and subsequent experimental lung metastasis was investigated in mice given GT-catechins. The accumulation at 6 and 24 h after injection of K1735M2 cells was significantly suppressed, and the number of lung-metastatic colonies was significantly reduced, in comparison with those in control mice. The results suggest that GT-catechin intake prevented the experimental tumor metastasis in aged SAMP10 mice via its inhibition of a reduction in immune surveillance potential with age.

Polychlorinated Biphenyls Disrupt Blood–Brain Barrier Integrity and Promote Brain Metastasis Formation

BackgroundPolychlorinated biphenyls (PCBs) comprise a ubiquitous class of toxic substances associated with carcinogenic and tumor-promoting effects as well as neurotoxic properties in the brain. However, the effects of PCBs on the development of tumor metastases are not fully understood.ObjectiveWe evaluated the hypothesis that exposure to individual PCB congeners can facilitate the development of brain metastases in immunocompetent mice via the disruption of the integrity of the blood–brain barrier (BBB).MethodsC57/Bl6 mice were exposed to individual PCBs by oral gavage, and 48 hr later they were injected with luciferase-labeled K1735 M2 melanoma cells into the internal carotid artery. The development of metastatic nodules was monitored by bioluminescent imaging. In addition, we evaluated the functional permeability of the BBB by measuring permeability of sodium fluorescein across the brain microvessels. Expression and colocalization of tight junction (TJ) proteins were studied by Western blotting and immunofluorescence microscopy.ResultsOral administration of coplanar PCB126, mono-ortho-substituted PCB118, and non-coplanar PCB153 (each at 150 μmol/kg body weight) differentially altered expression of the TJ proteins claudin-5, occludin, and zonula occludens-1 in brain capillaries. These alterations were associated with increased permeability of the BBB. Most importantly, exposure to individual PCB congeners enhanced the rate of formation and progression of brain metastases of luciferase-tagged melanoma cells.ConclusionsOur results show for the first time that exposure to individual PCBs can facilitate the formation of bloodborne metastases via alterations of the integrity of the brain capillary endothelium.

Enhanced Experimental Tumor Metastasis with Age in Senescence-Accelerated Mouse

Tumor metastasis is affected by the host immune surveillance system. Since aging may attenuate the host immune potential, the experimental tumor metastasis may be enhanced with age. In the present study, we investigated this alteration of experimental tumor metastasis with age. We used senescence-accelerated mice prone 10 (SAMP10) as a model of aged animals. Natural killer cell (NK) activity, as an indicator of immune surveillance potential, in 8-month-old (aged) SAMP10 mice was observed to be much lower than that in 2-month-old (young) mice. When we examined the in vivo trafficking of lung-metastatic K1735M2 melanoma cells in SAMP10 with positron emission tomography (PET), K1735M2 cells labeled with [2-18F]2-deoxy-2-fluoro-D-glucose ([18F]FDG) were observed in both young and aged SAMP10 just after injection of the cells, whereas the clearance of 18F from the lungs was retarded in aged animals. The accumulation of 5-[125 I]iodo-2'-deoxyuridine ([125 I]IUdR)-labeled K1735M2 cells in the lungs of SAMP10 at 24 h after injection was significantly higher in aged mice. Corresponding to these results, the number of metastatic colonies in the lung was larger in the aged SAMP10 of the experimental tumor metastasis model. The present study demonstrated that the aging process produced a susceptible environment allowing the tumor cells to metastasize due to decrease in the host immune surveillance potential with age.

Vascularization of Melanoma by Mobilization and Remodeling of Preexisting Latent Vessels to Patency

Tumors must manipulate the host vasculature to provide a blood supply adequate for their proliferation. Although tumors may arise as avascular masses, there is increasing evidence that some tumors begin to proliferate by first co-opting preexisting host blood vessels. By fluorescent vascular imaging, we provide evidence that the vasculature in orthotopically implanted melanoma arises from a preexisting red cell-deficient vascular network that remodels to patency to accommodate the requirements of the expanding tumor mass. Topical application of vascular endothelial growth factor to vascular beds generated immediate and robust vascular transitions that were morphologically similar to tumor-induced transitions. N(phi)-nitro-L-arginine, a nitric oxide inhibitor, significantly inhibited the growth of a syngeneic K1735M2 melanoma by reducing blood supply to the tumor by a mechanism independent of endothelial cell proliferation. These findings suggest that tumor-induced remodeling of red cell-deficient vessels to patency contributes to tumor vascularization and growth.

Specific immunotherapy against occult cancer metastases.

We investigated the efficacy of a preparation containing High Five (H5) insect cells infected with recombinant baculovirus encoding the murine interferon-beta gene (H5BVIFN-beta) against established primary tumors and occult lung metastases. Injection of live or lyophilized H5BVIFN-beta into established subcutaneous tumors of the highly metastatic murine UV-2237m fibrosarcoma or K-1735M2 melanoma in syngeneic mice eradicated both primary tumors and preexisting lung metastases. The therapeutic effects of H5BVIFN-beta were not observed in nude mice and were diminished in syngeneic mice depleted of CD4(+) and CD8(+) T cells. Immunohistochemical staining showed that tumors injected with H5BVIFN-beta were densely infiltrated by CD4(+) and CD8(+) T cells in mice with normal CD4/CD8 complement. These data demonstrate that, unlike most immunologic approaches in which prophylaxis can be achieved but eradication of established tumor is rare, lyophilized preparations of H5BVIFN-beta can serve as a novel immunotherapy against both primary tumors and their occult metastases.

Cell Adhesion Molecules and Cancer Metastasis

The adhesive interaction between tumor cells and host cells or the extracellular matrix plays a crucial role in metastasis formation. Therefore, understanding the mechanism controlling metastasis may assist in the development of antimetastatic therapy. We have used synthetic or recombinant polypeptide analogues containing the Arg-Gly-Asp (RGD) sequence found in the functional domains of fibronectin, such as poly(RGD) or CH-271, to regulate the mechanisms involved in cell adhesion during the metastatic process. Poly(RGD) inhibited experimental lung and liver metastasis effectively when coinjected i.v. with various types of tumors. In a model of spontaneous lung metastasis using the B16-BL6 melanoma, repeated administration of this polypeptide before or after surgical excision of the primary tumor resulted in a significant inhibition of tumor metastasis without affecting the growth of the primary tumor and substantially prolonged the survival time of mice. The mechanism responsible for the inhibition of tumor metastasis by the polypeptides is at least partly associated with the ability to interfere with cellular functions such as adhesiveness, motility and invasiveness in the process of metastasis. Combined treatment of the CH-271 fusion polypeptide and anticancer drugs, i.e., anti-adhesion therapy combined with chemotherapy, caused a marked inhibition of lung and liver metastasis of tumors as compared with either treatment alone or with the control. In contrast, the promotion of tumor cell interaction with immune cells via cell adhesion molecules, which differs from the anti-adhesive mechanism, may lead to the induction of anti-tumor immune responses and, consequently, to the inhibition of tumor metastasis. The transfection of the gene of the B7-1 adhesion molecule into tumor cells (B16-BL6 or K1735-M2 melanoma) resulted in the remarkable reduction of lung metastasis caused by the i.v. injection into mice. Immunization of B7-transfected tumor was effective as a tumor vaccine for preventing the metastasis of B7 negative original tumor cells. Thus, the regulation of the adhesive interaction with tumor cells may provide a new and promising approach for the control and prevention of cancer metastasis.

Antitumor activity of a novel podophyllotoxin derivative (TOP-53) against lung cancer and lung metastatic cancer: Utsugi T, Shibata J, Sugimoto Y et al. Taiho Pharmaceutical Co., Ltd., Hanno Research Center, Hanno, Saitama 357. Cancer Res 1996;56:2809–2814

We synthesized a potent new antitumor podophyllotoxin derivative (4beta-aminoalkyl-4'-O-demethyl-4-desoxypodophyllotoxin; TOP-53) in our search for a drug that has strong activity against lung cancer and lung metastatic cancer. TOP-53 exhibited twice the inhibitory activity of etoposide (VP-16) against topoisomerase II and induced DNA strand breaks but showed no inhibitory activity against tubulin polymerization. The in vitro cytotoxic activity of TOP-53 assessed as IC50 was 0.016-0.37 microg/ml and 0.26-8.9 microg/ml against marine tumor and human non-small cell lung cancer (NSCLC) cell lines, respectively. TOP-53 exerted significant efficacy equivalent to that of VP-16 on s.c.-implanted murine solid tumors (Colon 26, B16-BL6, and Lewis lung carcinoma) at doses 3-5 times lower than that of VP-16. In human tumor xenografts using NSCLC, TOP-53 was active for four of five tumors, whereas VP-16 was active for two of five tumors. Potent inhibitory activity of TOP-53 was also found against a lung tumor (Lewis lung carcinoma) and four lung metastatic tumors (NL-22 and NL-17 colon cancer, UV2237M fibrosarcoma, and K1735M2 melanoma). TOP-53 appeared to be more active against four of them than VP-16. Thus, TOP-53 is not only active against s.c.-implanted lung cancers but also strongly active against lung localized tumor and metastatic tumors in the lungs. The high selectivity of TOP-53 was attributed to its high distribution into the lung and its persistence. TOP-53 is expected to be highly effective against lung cancer including NSCLC and various lung metastatic tumors in the clinical field.

Differential effects of synthetic sphingosine derivatives on melanoma cell motility, growth, adhesion and invasion in vitro.

Cancer cell surface glycosphingolipids are considered to play a critical role in tumor growth and metastasis. However, the implications of glycoconjugates in the control of cell motility, which is considered to be involved in tumor invasion, are not fully understood. In this study, the effects of a series of synthetic sphingosine derivatives, obtained by the chemical transformation of azidosphingosines, on directional migration of K1735-M2 melanoma cells grown on type I collagen-coated surfaces were investigated. Following the application of 60 microM (2R, 3S, 4E)-2, 3-epimino-4-octadecen-3-ol (S4) the migration rate was 94 +/- 10 microns/day, compared with 377 +/- 22 microns/day in the control experiment. Six other analogues were not as potent. S4 also considerably down-modulated melanoma single cell motility. Inhibition of motile activity was associated with changes in the actin filament organization as well as with changes in the number and distribution of vinculin plaques. Moreover, the compound reduced the attachment abilities of melanoma cells to basement membrane Matrigel. Tumor cell invasion, however, was less affected and proliferation remained unimpaired after treatment with S4. These data suggest at least one intracellular mode of action of this particular synthetic sphingosine derivative by modulation of cytoskeletal organization. Melanoma cell motility and growth may be controlled independently via glycosphingolipids.

Vaccination of tumor cells transfected with the B7-1 (CD80) gene induces the anti-metastatic effect and tumor immunity in mice

The present study demonstrates that the transfection of B7-1 or its variant MB7-2 genes into MHC class I+ tumor cells (B16-BL6 or K1735-M2 melanoma) resulted in the remarkable reduction of lung metastasis caused by i.v. injection into immunocompetent syngeneic mice. However, i.v. injection of the transfectants into T cell-deficient nude mice did not affect reduction of lung tumor colonies as compared with parental wild-type tumors, suggesting that such an inhibitory effect was closely associated with T cell-mediated responses. The reduced metastasis of B7+ tumor cells consequently led to the significant prolongation of survival. Expression of B7 on tumor cells did not influence the tumorigenicity in vivo and tumor cell invasion into basement membrane Matrigel in vitro. We also found that immunization of X-irradiated B7 transfectants was effective as a tumor vaccine for preventing lung metastasis caused by i.v. injection of B7- parental B16-BL6 cells but not against other syngeneic 3LL tumors. Thus, the B7-mediated anti-metastatic effect was tumor-specific. Vaccinations of irradiated B7+ tumor cells before and after surgical excision of the s.c. inoculated primary B7- tumors on day 21 achieved effectively the prevention of spontaneous lung metastasis. Our report that vaccination of irradiated B7+ tumor cells led to a therapeutic effect in an established tumor metastasis model clearly expands and confirms previous related observations.

Costimulation by CD48 and B7-1 induces immunity against poorly immunogenic tumors.

Genetic modification of many types of mouse tumors to express the B7-1 or B7-2 molecules, natural ligands for the T cell-costimulatory molecule CD28, increases their immunogenicity. However, even after transfection with the B7-1 and/or B7-2 genes, poorly immunogenic tumors fail to elicit and efficient immune response. We report here that two such tumors, the Ag104A sarcoma and the K1735-M2 melanoma, become immunogenic after transfection of the genes encoding murine B7-1 together with CD48, which is the natural ligand for CD2. Tumor-specific CD8+ cytotoxic T lymphocytes were readily generated and were effective for adoptive immunotherapy of metastasis induced by wild-type Ag104A sarcoma cells. A similar approach may be useful for developing therapy for other poorly immunogenic tumors, including those in humans.

Control of Integrin Expression by Extracellular Matrix

Integrin-mediated interactions between cells and the extracellular matrix play a fundamental role in the development and function of a variety of tissues by triggering intracellular signals that regulate gene expression. In this study, mouse mammary epithelial cells plated on tissue culture plastic were shown to dramatically up-regulate the steady state levels of mRNA encoding the alpha 1, alpha 2, alpha 3, alpha 5, alpha 6, alpha 7, alpha v, and beta 1 integrin subunits, in contrast to cells cultured on a basement membrane matrix or cells in vivo. This pattern of expression was also observed in a mouse mammary epithelial strain, CID-9 and in other mouse cell lines such as MMTE cells and K1735-M2 melanoma cells. The control of integrin expression was mediated at different levels in different cell types. In K1735-M2 cells, transcription of the beta 1 integrin gene was influenced by the substratum, although the levels of integrin protein remained similar. In mammary epithelial cells, the rates of beta 1 integrin gene transcription were similar, but mRNA and protein levels were higher in cells cultured on plastic than those on basement membrane. For both cell types, the rate of integrin protein turnover was nearly identical in cells cultured on either substratum. Our results demonstrate that extracellular matrix controls the expression of beta 1 integrin subunits and that this regulation is exerted at both transcriptional and post-transcriptional levels.

Inhibition of K1735-M2 melanoma cell invasionin vitro by retinoic acid

Inhibition of K1735-M2 melanoma cell invasionin vitro by retinoic acid

Inhibition of K1735-M2 melanoma cell invasion in vitro by retinoic acid.

Melanoma cell invasion in vitro was tested by means of confrontation cultures of melanoma multicellular spheroids with rounded fragments of embryonic chick heart tissue. Quantitative determination of invasion was performed using a computerized image analysis program, facilitating the evaluation of the efficacy of potentially anti-invasive compounds. Retinoic acid (RA; 1 microM) [corrected] considerably impaired K1735-M2 melanoma cell invasion, as demonstrated by various measuring parameters. Parameter TUMAREA, expressing the amount of tumor tissue, indicates a growth inhibitory effect and the invasion parameter STRCSTR shows that after treatment with RA the stromal component was better preserved than in untreated controls. Besides the inhibitory effect of RA on melanoma cell invasion in confrontation cultures, RA increased the dynamics of adhesion of melanoma cells to the extracellular matrix components type I collagen and laminin, and slightly impaired melanoma cell directional migration. Fluorescence microscopy using rhodamine-labeled phalloidin showed that RA also modulated the organization of the actin cytoskeleton by inducing the formation of actin-containing stress fibers. Our data show that 1 microM RA exhibited a pronounced anti-invasive effect on highly metastatic melanoma cells in vitro. Impairment of host tissue degradation, altered adhesion abilities, changes in the actin cytoskeleton, as well as the antiproliferative effect may all account for inhibition of melanoma cell invasion.

Inhibition of melanoma cell directional migration in vitro via different cellular targets

In malignant melanoma active movement of cancer cells is considered to be essential for tissue invasion. Various mechanisms, such as the Ca(2+)-calmodulin-proteinkinase C cascade or G-protein-dependent processes are considered to play a role in tumor cell functions. The assay of directional migration, combined with computer-assisted image analysis, was used to evaluate the antimigratory efficacy of drugs interfering with different steps of signal transduction pathways. Treatment with different compounds showed a more or less concentration-dependent reduction of migration rates: The Ca(2+)-channel blockers verapamil and devapamil showed a slight reduction of motility. The effect was more pronounced when the calmodulin antagonist flunarizine was used or the proteinkinase C inhibitors dequalinium, tamoxifen and H-7 were applied. A marked inhibition of motility was found with the G-protein antagonist L 651582. Thus, our results indicate that different signal transduction pathways are involved in the regulation of directional migration of K1735-M2 melanoma cells.

Effect of dequalinium on K1735-M2 melanoma cell growth, directional migration and invasion in vitro

Cationic lipophilic compounds have an antiproliferative effect on certain tumour systems in vitro and in vivo. We have investigated whether the cationic lipophilic compound dequalinium affects not only proliferation but also motility and invasion of the highly metastatic and highly invasive melanoma cell line K1735-M2. Proliferation was assessed in monolayer cultures and in multicellular spheroids, motility was estimated in the assay of directional migration, and invasiveness was tested through confrontation cultures of tumour multicellular spheroids with embryonic chick heart tissue evaluated by computerized image analysis. 2 μmol/l dequalinium impaired melanoma cell proliferation, reduced directional migration and significantly blocked invasion in vitro. On the ultrastructural level, dequalinium caused obvious changes in mitochondria of both melanoma and embryonic chick heart cells. The mechanisms of the antiproliferative, antimigrating and antiinvasive effects remain to be determined. Inhibition of protein kinase C, calmodulin antagonism, DNA intercalation and/or direct effects on mitochondrial functions may be considered.