Natural product pectolinarigenin exhibits potent anti-metastatic activity in colorectal carcinoma cells in vitro and in vivo

Colorectal carcinoma (CRC) is one of the most common cancers with high metastatic potential, explaining why identifying new drug candidates that inhibit tumour metastasis is an urgent need. The aim of this study was to evaluate the biological activities of pectolinarigenin (PEC, a natural flavonoid present in Cirsium chanroenicum) in CRC in vitro and in vivo and to determine its underlying mechanism of action. Here, we observed that treatment with PEC could inhibit cell viability and induce apoptosis in cancer cells in a concentration- and time-dependent manner. The occurrence of apoptosis was associated with activation of caspase-3 and Bax and decreased expression of Bcl-2. In addition, PEC markedly impaired CRC cell migration and invasion by downregulating the expression of matrix metalloproteinase (MMP-9) and phosphorylated-Stat3^Tyr705. Moreover, our studies showed that PEC inhibited abdominal metastasis models of murine colorectal cancer. In addition, histological and immunohistochemical analyses revealed a decrease in Ki67-positive cells, MMP9-positive cells and p-Stat3^Tyr705 cells upon treatment with PEC compared to control samples. Furthermore, PEC reduced the number of myeloid-derived suppressor cells (MDSCs) in the blood and tumours, which was accompanied by the increased infiltration of CD8⁺T cells in the blood. Taken together, our findings suggested that PEC could be used as a natural drug to inhibit CRC metastasis.     

Atrophy of compartments of rat lymph nodes related to an entry of lethally altered lymphocytes

Summary This paper reports the occurrence of an accumulation of lethally altered lymphocytes in the subcapsular sinus of a compartment or compartments of some lymph nodes, an unusual feature best developed in nodes of the mesenteric site in aging athymic animals. Many of these cells are rod-like. In other compartments, similar lymphocytes occurred at various depths in the nodal parenchyma. This was accompanied by the disappearance of a compartment's populations of normal lymphoid cells. The observations reveal that lymphocytes, altered in a tissue, may reach the subcapsular sinus of the draining node compartment and migrate into its parenchyma which then undergoes atrophy. The likely involvement of mast cells is discussed.This work was funded by the Medical Research Council of Canada.     

Microfibrils: a constitutive component of reticular fibers in the mouse lymph node

Summary Fibrous components other than collagen fibrils in the reticular fiber of mouse lymph node were studied by electron microscopy. Bundles of microfibrils not associated by elastin and single microfibrils dispersed among collagen fibrils were present. The diameter of the microfibrils was 13.29±2.43 nm (n=100). Elastin-associated microfibrils occurred at the periphery of the reticular fiber. Elastin was enclosed by microfibrils, thus forming the elastic fiber, which was clearly demonstrated by tannic acid-uranyl acetate staining. In the reticular fiber of lymph nodes, the elastic fiber consisted of many more microfibrils and a small amount of elastin. These microfibrils, together with the collagen fibrils, may contribut to the various functions of the reticular fibers.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

In situ activation of mouse macrophages and therapy of spontaneous renal cell cancer metastasis by liposomes containing the lipopeptide CGP 31362

Summary We determined whether the intravenous administration of multilamellar vesicle liposomes (MLV) containing a lipopeptide analogue of a fragment from the cell wall of gram-negative bacteria (CGP 31 362) can render BALB/c mouse alveolar macrophages tumoricidal in situ and reduce the incidence of spontaneous lung metastasis of syngeneic renal carcinoma (RENCA) cells. Alveolar macrophages (a) incubated in vitro with MLV containing CGP 31 362 (MLV-31 362) and (b) harvested from mice injected i.v. with MLV-31 362 were rendered cytotoxic against the RENCA cells. Maximum cytotoxic activity of the macrophages was induced by injecting 5 µmol MLV consisting of 250 mg phospholipids and 0.5 mg CGP 31 362. The single i.v. injection of 5 µmol MLV-31 362 produced activation of macrophages that lasted for up to 4 days. Repeated i. v. injections of MLV-31 362 produced a continuous antitumor activity in alveolar macrophages. To study the lipopeptide's effects on metastasis, we injected the left kidneys of BALB/c mice with RENCA cells. The kidney with growing tumor was resected 10 days later and, after a further 2 days, groups of mice were injected i.v. with MLV-31 362 or with MLV-HBSS (twice weekly for 3 weeks). Treatment with MLV-31 362 significantly decreased the median number of spontaneous lung metastases. These data demonstrate that the systemic administration of MLV-31 362 can activate murine lung macrophages in situ and reduce the incidence of spontaneous RENCA lung metastases.     

In situ activation of syngeneic tumour-specific cytotoxic T lymphocytes: Intra-pinna immunization followed by restimulation in the peritoneal cavity

Summary Tumour-specific cytotoxic T lymphocytes (CTL) are usually obtained after immunization in vivo and restimulation of immune cells in vitro. We here describe the generation of syngeneic tumour-specific CTL within no more than 9 days by priming and restimulation in vivo. This is achieved only if the correct sites are used both for primary immunization (ear pinna) and for restimulation (peritoneal cavity). The kinetics of immune T cell induction and of the secondary response in vivo will be reported. While a secondary CTL response could be generated in the peritoneal cavity, this was not possible in the spleen, no matter which routes of antigen restimulation were used. Upon transfer of immune spleen cells into the peritoneal cavity but not into the spleen, a secondary response could be generated upon in situ restimulation, indicating the importance of the correct microenvironment for this type of response. The peritoneal effector cells were true T cells and recognized a tumour-associated antigen in association with the K^d major histocompatibility (MHC class I) antigen. Finally the activated tumour-specific peritoneal exudate cells were able to transfer protective immunity without exogenous interleukin-2 into normal syngeneic mice.     

The early postnatal development of the primary immune response in TNP-KLH-stimulated popliteal lymph node in the rat

Summary The popliteal lymph nodes were removed from young rats of various ages five days after a single immunization with TNP-KLH in the hind footpads. Cryostat sections of the lymph nodes were investigated by means of enzyme and immunohistochemical techniques at the light-microscopical level.The presence and localization of anti-TNP antibody-containing cells were examined using a new technique to visualize specific antibodies. Moreover, the development of the lymph nodes following exogenous antigenic stimulation was compared with that of unstimulated lymph nodes.Specific antibody-containing cells could not be found before day 15 after birth, in rats immunized at day 10. From that time these lymphoid cells were located primarily at the border between cortex and medulla. Younger popliteal lymph nodes showed only aspecific immunoglobulin-containing lymphoid cells. With age, the number of specific antibody-containing cells tended to increase. These cells were more mature, according to morphological criteria and were located nearer the medulla.The first primary follicles were seen at day 19, as was the case in unstimulated animals. The first secondary follicles, containing germinal centers, were detected at day 23, whereas in unstimulated popliteal lymph nodes they were never found.Trapping of immune complexes could not be demonstrated before day 33 after birth. The later appearance of this phenomenon might be a consequence of the techniques applied to demonstrate specific antibody-containing cells.Abbreviations PLN popliteal lymph node - FDC follicular dendritic cell - IDC interdigitating cell - HEV high endothelial venule - TNP trinitrophenyl - KLH keyhole limpet hemocyanin - PBS phosphate-buffered saline - GCPC germinal center precursor cell - sIg surface immunoglobulin - cIg cytoplasmic immunoglobulin     

Carbohydrate-binding proteins of tumor lines with different growth properties

Summary A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weights of 80 kDa, 70 kDa, 22 kDa, 18 kDa and 16 kDa and of a fucose-binding protein at apparent molecular weight of 42 kDa. The -glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5–8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5–8#1 and 5–20#20, whereas the metastatic variant 5–20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.     

Biomechanical interactions of cancer cells with the microvasculature during metastasis

Metastasis is a major, life-threatening complication of cancer. The bloodstream is the most important disseminative route for cancer cells liberated from their parent tumors. Single circulating cancer cells are arrested in the microvasculature, where the vast majority are killed by rapid or slow processes, and the relatively few survivors grow into micrometastases. We review the underlying causes of one type of rapid cancer cell death in the microcirculation, namely, that caused by biomechanical interactions of cancer cells with microvessel walls, which may result in cell surface membrane expansion and lethal rupture. These lethal interactions appear to be important rate-regulators in hematogenous metastasis, and to dictate some aspects of metastatic patterns. Although these are not the only interactions involving cancer cells, in contrast to others involving cellular and humoral defense mechanisms, they have received comparatively little attention.     

Lymphocyte subpopulations of regional lymph nodes in human colon and gastric adenocarcinomas

 In order to study the host immune response to tumours, previous knowledge of the cellular composition of regional draining lymph nodes is necessary. Enlarged regional lymph nodes are a common finding in colon and gastric adenocarcinomas. We have studied the cellular composition of normal non-reactive and of regional draining lymph nodes of colon and gastric adenocarcinomas. In normal non-reactive lymph nodes, T lymphocytes (CD2⁺, CD7⁺) constituted the largest fraction of the lymphoreticular cells. These lymphocytes were mainly CD4⁺, and there were more cells expressing the CD45RA isoform of the CD45 antigen than CD45RO. Reactive lymph nodes presented a decreased proportion of CD4⁺ CD45RA⁺ cells and an increased number of B cells. Although most of the T cells in the reactive nodes were CD4⁺ CD45RO⁺, their proportion was similar to that found in normal non-reactive nodes. We studied the presence of the molecules CD28 and CD80 involved in the processes of interaction and activation of T and B lymphocytes. The CD28 molecule was found in all the T lymphocytes, while the CD80 molecule was weakly expressed on the B lymphocyte membrane. Received: 4 January 1996 / Accepted: 28 May 1996