Priming effect of aspirin for tumor cells to augment cytotoxic action of cisplatin against tumor cells: implication of altered constitution of tumor microenvironment, expression of cell cycle, apoptosis, and survival regulatory molecules

The present study was conducted to investigate if anti-inflammatory drug aspirin could alter the cytotoxic action of cisplatin on tumor cells. Using a transplantable T cell lymphoma in a murine model, we demonstrate that exposure to aspirin exerts a priming action on tumor cells, rendering them susceptible to induction of cell death by cisplatin with consequences on retardation of tumor progression. The priming action of aspirin on tumor cells was found to be dependent on an altered constitution of tumor microenvironment with respect to decline of acidosis and modulation in the expression of cell cycle and survival regulatory molecules like cyclin B1, cyclin D, bcl-2, bcl-xL, p53, and cytokines: IL-4, IL-10, IFN- γ & VEGF. The study also discusses possible mechanisms underlying augmentary action of aspirin on cisplatin-mediated tumor cells killing. This is the first report showing that pre-exposure of tumor cells to aspirin lowers the concentration of cisplatin to exert its cytotoxic action. The finding of this study will help in designing novel antitumor protocols with reduced dose of cisplatin.     

Antitumoral action of bovine seminal ribonuclease

Summary The antitumor action of bovine seminal RNAase is studied as a function of the enzyme concentration and of the number of plated cells. With polyoma transformed hamster kidney cells, a 50% inhibition of cell growth is obtained with a 10 μg/ml of enzyme, while at this concentration growth of normal cells is very little affected. On the other hand the higher the number of plated cells, the lesser is the effect. The enzyme is found to be very effective also on tumor cells derived from a spontaneous tumor (neuroblastoma) and on cells derived from a chemically induced tumor (glioma). AmphoterycinB which is known to alter the permeability of eukariotic cells, does not affect the resistance of normal cells to the cytotoxic action of the enzyme.     

Toxicity of para-aminobenzhydrazide and its influence on nucleic acid biosynthesis in cultures of normal and tumor cells

We have investigated cytotoxic action of p-aminobenzhydrazide and its influence on biosynthesis of nucleic acids in cultures of intact cells, tumor cells and intact cells stimulated by phytohemagglutinin. p-Aminobenzhydrazide is considered as a representative of hydrazine's derivatives (in particular, of hydrazine sulphate). We compare its action with that of a typical cytotoxic agent such as iododeoxyuridine. We have found that p-aminobenzhydrazide influences biosynthesis of nucleic acids in the same way as iododeoxyuridine. However it acts toxically on tumor cells though it is not toxic for intact cells so that its action is different as compared to that of cytotoxic agents. Specific toxic action of aminobenzhydrazide on tumor cells may be due to the enhancement of antitumor activity substances of this compound and absence of such enhancement of side toxic effects.     

Involvement of substance P and the NK-1 receptor in cancer progression

Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis.     

Effect of liposomal antitumor preparation 5,6-benzocoumarin-5-uracil on intensity of free-radical processes in the liver microsomes and tumor cells of rats with transplanted Guerin's carcinoma

The contents of primary and secondary (TBA-active) products of lipid peroxidation were investigated in microsomal fraction of the liver and tumor cells of rats with transplanted Guerin's carcinoma and under the condition of antitumor liposomal preparation 5,6-benzcumarine-5-uracil (BCU) action. High level of lipid peroxidation process in the microsomal fraction is shown in the rat liver and tumor cells under the condition of BCU action in the period of intensive carcinoma growth. It remains till the period of tumor growth braking. This fact testifies to the prooxidation action of the preparation. Liposomal antitumor preparation BCU raises the process of lipid peroxidation in microsomal fraction of tumor cells and its action increases according to the malignant growth. The processes of lipid peroxidation in microsomal rat liver fraction approach the control data under the condition of the mentioned preparation. The investigated liposomal form of BCU possesses directed prooxidation action on the malignant tissue.     

Oncostatic action of melatonin: facts and question marks

The paper presents the data concerning the in vivo effects of melatonin on experimentally-induced tumors in animals and the in vitro effects on animal and human tumor cells. The majority of experimental tumors responded to the melatonin treatment with growth inhibition. However, some negative or opposite results (i.e. stimulation of tumor instead of inhibition) were also reported. Some of the negative results can be attributed to the improper timing of melatonin administration. Melatonin was also shown to inhibit the growth of several animal and human tumor cell lines in vitro. On the basis of these experiments, a hypothesis of the oncostatic action of melatonin was put forward. The mechanism of the postulated action is complex and probably includes: 1) modulation of the endocrine system; 2) modulation of the immune system; 3) the direct oncostatic action of melatonin on tumor cells. The latter includes the recently discovered anti-oxidative action which probably plays an important role in the countering the DNA damage during the radiation challenge or the exposure to chemical carcinogens. It also includes the antiproliferative and pro-apoptotic effects exerted via melatonin receptors expressed by tumor cells. The involvement of the membrane melatonin receptors is mainly assumed. However, the recent data from our and other laboratories suggest also the involvement of RZR/ROR receptors (the putative melatonin nuclear receptors) in both melatonin-induced proliferation inhibition and apoptosis.     

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.     

Antiapoptotic Factor Humanin Is Expressed in Normal and Tumoral Pituitary Cells and Protects Them from TNF-α-Induced Apoptosis

Humanin (HN) is a 24-amino acid peptide with cytoprotective action in several cell types such as neurons and testicular germ cells. Rattin (HNr), a homologous peptide of HN expressed in several adult rat tissues, also has antiapoptotic action. In the present work, we demonstrated by immunocytochemical analysis and flow cytometry the expression of HNr in the anterior pituitary of female and male adult rats as well as in pituitary tumor GH3 cells. HNr was localized in lactotropes and somatotropes. The expression of HNr was lower in females than in males, and was inhibited by estrogens in pituitary cells from both ovariectomized female and orquidectomized male rats. However, the expression of HNr in pituitary tumor cells was not regulated by estrogens. We also evaluated HN action on the proapoptotic effect of TNF-α in anterior pituitary cells assessed by the TUNEL method. HN (5 µM) per se did not modify basal apoptosis of anterior pituitary cells but completely blocked the proapoptotic effect of TNF-α in total anterior pituitary cells, lactotropes and somatotropes from both female and male rats. Also, HN inhibited the apoptotic effect of TNF-α on pituitary tumor cells. In summary, our results demonstrate that HNr is present in the anterior pituitary gland, its expression showing sexual dimorphism, which suggests that gonadal steroids may be involved in the regulation of HNr expression in this gland. Antiapoptotic action of HN in anterior pituitary cells suggests that this peptide could be involved in the homeostasis of this gland. HNr is present and functional in GH3 cells, but it lacks regulation by estrogens, suggesting that HN could participate in the pathogenesis of pituitary tumors.     

The importance of G0 in the site of action of interferon in the cell cycle

It has been suggested that the ‘metabolic event’ controlling the transition of quiescent cells into the proliferating phase is a possible target for the growth inhibitory action of interferon [1]. Direct evidence of this mechanism of action has been hindered by the lack of techniques for the quantification of quiescent cells in a tumor population. The present report applies a recently available technique [2] to investigate more closely the site of interferon action in the G1 part of the cell cycle, and to emphasize that a G0 population is a necessary but not sufficient determinant of a cell line's response to growth inhibition by interferon.     

A proapoptotic peptide conjugated to penetratin selectively inhibits tumor cell growth

The peptide KLA (acetyl-(KLAKLAK)₂-NH₂), which is rather non toxic for eukaryotic cell lines, becomes active when coupled to the cell penetrating peptide, penetratin (Pen), by a disulfide bridge. Remarkably, the conjugate KLA–Pen is cytotoxic, at low micromolar concentrations, against a panel of seven human tumor cell lines of various tissue origins, including cells resistant to conventional chemotherapy agents but not to normal human cell lines. Live microscopy on cells possessing fluorescent labeled mitochondria shows that in tumor cells, KLA–Pen had a strong impact on mitochondria tubular organization instantly resulting in their aggregation, while the unconjugated KLA and pen peptides had no effect. But, mitochondria in various normal cells were not affected by KLA–Pen. The interaction with membrane models of KLA–Pen, KLA and penetratin were studied using dynamic light scattering, calorimetry, plasmon resonance, circular dichroism and ATR-FTIR to unveil the mode of action of the conjugate. To understand the selectivity of the conjugate towards tumor cell lines and its action on mitochondria, lipid model systems composed of zwitterionic lipids were used as mimics of normal cell membranes and anionic lipids as mimics of tumor cell and mitochondria membrane. A very distinct mode of interaction with the two model systems was observed. KLA–Pen may exert its deleterious and selective action on cancer cells by the formation of pores with an oblique membrane orientation and establishment of important hydrophobic interactions. These results suggest that KLA–Pen could be a lead compound for the design of cancer therapeutics.     

Gender dimorphism of tumor growth: role of gonadal hormones in differential regulation of apoptosis of a murine T cell lymphoma

The present investigation was undertaken to study if a gender-dependent differential induction of tumor cell apoptosis is responsible for the manifestation of gender dimorphism observed in the growth of a transplantable murine T cell lymphoma, designated as Dalton’s lymphoma (DL). Tumor cell samples obtained from male tumor-bearing mice showed a higher number of cells with apoptotic morphology compared to that observed in female tumor-bearing mice. In this report we demonstrate that male hormone androgen and female hormone estrogen can differentially modulate tumor cell proliferation and apoptosis through alteration in the expression pattern of cell death regulating genes: p53 and CAD. DL cells were shown to express mRNA for androgen and estrogen receptors. Further these gonadal hormones also induced tumor cells to produce tumor growth regulating proteins: VEGF, TGF-β, IL-2, IL-2R, SOCS, Hsp-70 and IFN-γ which in turn either through autocrine action on tumor cells or via TAM-derived NO were observed to regulate tumor cell apoptosis leading to gender dimorphism of tumor growth. This study also discusses the possible mechanism involved. The study has clinical significance as these results will helps in understanding the mechanism of gender dimorphism with respect to the progression of T-cells tumors.     

RELATIONSHIP OF NUTRITIONAL FACTORS TO IN VITRO TUMOR CELL GROWTH AND CYTOTOXICITY PRODUCED BY CYTOSINE ARABINOSIDE

The in vitro relationship between nutritional factors, proliferative status of tumor cells, and the cytotoxic action of cytosine arabinoside (ara-C) was investigated. The reduction in the concentration of only one essential amino acid, L-isoleucine, in the growth medium of A(T₁)Cl-3 hamster fibrosarcoma cells decreased DNA synthesis in this cell population and slowed the rate of progression of G₁ phase cells into S phase of the cell cycle. The complete omission of isoleucine from the growth medium blocked the progression of G₁ phase cells into S phase and prevented the cytotoxic action of ara-C. The addition of isoleucine to the isoleucine-deprived cells permitted these cells to enter the S phase and restored their sensitivity to the cytotoxic action of ara-C. When G₁ phase cells were placed in a medium containing reduced levels of all the amino acids and vitamins there was a prolongation of the G₁ phase. Since medium with low levels of amino acids produced a delay in the entry of G₁ phase cells into the S phase, the time interval in which these cells were most sensitive to the cytotoxic action of ara-C was different for G₁ phase cells placed in medium with adequate levels of all the amino acids. These in vitro data indicate that nutritional factors can markedly effect the proliferation of tumor cells and the cytotoxic action of ara-C.     

The many faces of calmodulin in cell proliferation,programmed cell death,autophagy, and cancer

Calmodulin (CaM) is a ubiquitous Ca^2 + receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.     

Antitumor effect of arabinogalactan and platinum complex

The article presents the results of investigation of antitumor properties of platinum–arabinogalactan complex. We showed the ability of the complex to inhibit the growth of Ehrlich ascites tumor cells. It is found that the distribution of the platinum–arabinogalactan complex is not specific only for tumor cells in mice. The complex was found in all tissues and organs examined (ascites cells, embryonic cells, kidney, and liver). The mechanism of action of the arabinogalactan–platinum complex may be similar to cisplatin as the complex is able to accumulate in tumor cells.     

Cytotoxic activity of a tumor protease-activated pore-forming toxin

Equinatoxin II is a pore forming toxin produced by the sea anemone Actinia equina. It is able to kill very unspecifically most cell types by the membrane-perturbing action of an amphiphilic alpha-helix located at its N-terminal. A normally active N-terminal mutant, containing one single cys in the amphiphilic alpha-helix, becomes totally inactive when it is bound to avidin via a biotinylated linker. By choosing, as a linker, a peptide containing a tumor protease cleavage site, we were able to construct an enzymatically activable conjugate which should be selective for tumor cells. The introduced cleavage site was designed in order to be digested by both cathepsin B and matrix metalloproteases (MMPs). We confirmed that this conjugate could be activated in vitro by cathepsin B and MMPs. After having measured the enzymatic activity of fibrosarcoma and breast carcinoma cells, we analyzed the cytotoxic effect of the conjugate on the same lines and on human red blood cells (HRBC) as controls. We found that the conjugate was activated, at least in part, by the tumor cell lines used, whereas it was inactive on HRBC. That the activation process was dependent on the enzymatic action of cathepsin B and MMPs, was indicated by three lines of evidence: (1) binding occurred normally on all type of cells including HRBC which however were insensitive being devoid of enzymes; (2) the cytotoxic effect correlated with the amount of cathepsin B activity expressed by the cells; (3) conjugate activation was reduced by specific inhibitors of cathepsin B and MMPs. These results demonstrate the possibility of tumor cell killing by a pore-forming toxin conjugate specifically activated by tumor proteases.     

Macrophage-mediated cytostasis of neoplastic hemopoietic cells: cytofluorometric analysis of the reversible cell cycle block

Nonactivated mouse peritoneal macrophages inhibit the proliferation of neoplastic hemopoietic cells in vitro. This effect is dependent upon the number of adherent macrophages present in cultures of hemopoietic tumor cells and can be documented by various parameters used as indices of cell proliferation. The two-layer soft agar culture system permits analysis of the regulatory functions of macrophage-derived diffusible substances under conditions of extremely low cell density and where macrophage-tumor cell contact is prevented by the gel matrix. The ability of the underlayers of macrophages to inhibit colony formation by hemopoietic tumor cells indicates that such an effect can be mediated by factor(s) elaborated by macrophages. Evidence that the mode of action of macrophages on tumor cell proliferation is cytostatic rather than cytotoxic is the capability of the macrophages to retard tumor cell growth in a particular phase of the cell cycle with retention of cell viability. Growth inhibition could be demonstrated to be reversible, with tumor cells entering normal cell cycle distributions shortly after being removed from macrophages or macrophage-derived factors. Cytofluorometric analysis of cell cycle inhibition correlated with viable cell counts and mitotic indices and confirms the suitability of this method for studying tumor cell proliferation.     

Humanin inhibits apoptosis in pituitary tumor cells through several signaling pathways including NF-κB activation

Humanin (HN) and Rattin (HNr), its homologous in the rat, are peptides with cytoprotective action in several cell types such as neurons, lymphocytes and testicular germ cells. Previously, we have shown that HNr is expressed in pituitary cells and that HN inhibited the apoptotic effect of TNF-α in both normal and tumor pituitary cells. The aim of the present study was to identify signaling pathways that mediate the antiapoptotic effect of HN in anterior pituitary cells from ovariectomized rats and in GH3 cells, a somatolactotrope cell line. We assessed the role of STAT3, JNK, Akt and MAPKs as well as proteins of the Bcl-2 family, previously implicated in the antiapoptotic effect of HN. We also evaluated the participation of NF-κB in the antiapoptotic action of HN. STAT3 inhibition reversed the inhibitory effect of HN on TNF-α-induced apoptosis in normal and pituitary tumor cells, indicating that STAT3 signaling pathway mediates the antiapoptotic effect of HN on pituitary cells. Inhibition of NF-κB pathway did not affect action of HN on normal anterior pituitary cells but blocked the cytoprotective effect of HN on TNF-α-induced apoptosis of GH3 cells, suggesting that the NF-κB pathway is involved in HN action in tumor pituitary cells. HN also induced NF-κB-p65 nuclear translocation in these cells. In pituitary tumor cells, JNK and MEK inhibitors also impaired HN cytoprotective action. In addition, HN increased Bcl-2 expression and decreased Bax mitochondrial translocation. Since HN expression in GH3 cells is higher than in normal pituitary cells, we may suggest that through multiple pathways HN could be involved in pituitary tumorigenesis.     

The cryopreservation of immunocompetent cells

Lymphoid cells sensitized against tumor cells in vivo were removed from sheep and mice, frozen and thawed, and then assayed for retention of cytotoxic function. Sheep lymphocytes efferent from immunized nodes have been shown previously to be cytotoxic owing to the production of antibody and these cells retained approximately 50% of their activity after freezing. The specific cytotoxicity of sensitized mouse spleen cells has been shown to be due to thymus-derived lymphocytes and such cells totally retained specific cytotoxic action after freezing. However, spleen cells also generally exhibited a degree of nonspecific cytotoxic action which disappeared on freezing and was attributed to the selective elimination of glass-adherent cells by the freezing process.     

Eicosapentaenoic acid attenuates vascular endothelial growth factor-induced proliferation via inhibiting Flk-1 receptor expression in bovine carotid artery endothelial cells

Eicosapentaenoic acid (EPA; 20:5, n-3) can restrain tumor growth and metastasis in vivo; however, the mechanism of its antitumor effect is still not fully understood. Angiogenesis is a crucial process for tumor growth and metastasis and inhibition of tumor angiogenesis can suppress tumor growth and metastasis in vivo. Vascular endothelial growth factor (VEGF) is an important angiogenic factor. In this study, we investigated the mechanisms of the inhibitory effect of EPA on VEGF-induced proliferation of bovine carotid artery endothelial (BAE) cells. BAE cells, treated with 0–5 μg/ml EPA for 48 h, displayed a dose-dependent suppression to VEGF (0.2 nM)-induced proliferation. Similar inhibitory effect was not found in BAE cells treated with arachidonic acid (AA; 20:4, n-6), or docasahexaenoic acid (DHA; 22:5, n-3). In contrast to its effect on VEGF-induced proliferation, EPA had no inhibition to basic fibroblast growth factor (bFGF, 0.2 nM)-induced proliferation in BAE cells. Both VEGF and bFGF activated mitogen-activated protein (MAP) kinase in BAE cells; however, EPA selectively inhibited VEGF-induced, but not bFGF-induced activation of MAP kinase. Flk-1 expression was inhibited dose-dependently in EPA-treated cells, whereas Flt-1 expression was increased in EPA treated cells. This in vitro inhibitory effect by EPA on Flk-1 receptor expression provides indirect evidence that one of the mechanisms of EPA for antitumor action in vivo maybe related to its antiangiogenic action. J. Cell. Physiol. 176:342–349, 1998. © 1998 Wiley-Liss, Inc.