Lyophilized whole human melanoma cells stimulate human PBMC proliferation and enhance suppressive action of PBMC toward survival of the same malignant cell line in vitro.

The goal of this work was to determine: a) do lyophilized human melanoma BG or Fem-X cells affect the proliferative capacity of normal human peripheral blood mononuclear cells (PBMC) and b) does the PBMC six-days preincubation in nutrient medium with FBS with, or without lyophilized human melanoma BG or Fem-x cells, affect their suppressive action on the survival of the same malignant cell line in vitro. In the aim to avoid any stimulating effect of FBS, other group of experiments were done in nutrient medium with human AB serum in order to determine: c) does the PBMC six-day-preincubation with lyophilized human melanoma BG or Fem-x cells affect their antiproliferative action on the corresponding malignant cell line in vitro and d) does the PBMC six-day preincubation with lyophilized normal PBMC, obtained from healthy volunteer (as a source of allogenous, but not of tumor antigens), affect their suppressive action on the survival of both melanoma BG and Fern-x cell lines in vitro. Results obtained in the presence of FBS in nutrient medium, showed that lyophilized BG cells induced a proliferation of the healthy PBMC, depending on the number of stimulating lyophilized cells. Lyophilized Fem-x cells induced healthy PBMC proliferation in lesser degree than lyophilized BG cells. This stimulation was almost constant, not dependent on the number of stimulating lyophilized Fem-x cells. Six-day stimulation in vitro by both lyophilized melanoma cells enhanced the suppressive action of PBMC on the survival of the corresponding malignant cell line. Experiments done in nutrient medium with normal human AB serum showed that six-day stimulation with lyophilized melanoma cells enhanced, again, the suppressive action of PBMC on the survival of the corresponding malignant cell line. Contrary, six day preincubation of normal PBMC with the lyophilized healthy PBMC (obtained from other healthy person) inhibited their suppressive action on the survival of both malignant cell lines in vitro.

Antiproliferative action of isatine-beta-thiocarbohydrazone and N-ethylisatine-beta-thiocarbohydrazone on human PBMC and on two neoplastic cell lines.

The antiproliferative action of two synthetic compounds, isatine-b-thiocarbohydrazone (IsTCH) and N-ethyl isatine-beta-thiocarbohydrazone (N-Et-IsTCH), towards healthy human peripheral blood mononuclear cells (PBMC) and two neoplastic cell lines in vitro, was investigated. IsTCH and N-Et-IsTCH were dissolved in DMSO and then diluted with nutrient medium to desired final concentration. Target cells were PBMC, as well as human cervix carcinoma - HeLa cells, and murine melanoma B16 cells. Five different concentrations (3 microM to 50 microM) of investigated agents were applied on target cells. Cell survival was determined 72 h after the agent's action using MTT test. Results obtained showed that both investigated compounds exerted a dose dependent antiproliferative action to neoplastic cell lines. Their action was only cytostatic; trypan blue exclusion test did not show any sign of direct drug cytotoxicity when drugs concentration were less than 50 microM. ICs50 +/- SD for IsTCH antiproliferative action were 61.69 +/- 4.25 microM for HeLa cells; 34.1 +/- 7.15 microM for B16 cells: 17.62 +/- 7.11 microM for nonstimulated and 30.0 +/- 9.46 microM for stimulated (by 5 mg/ml PHA) PBMC. ICs50 +/- SD for the action of N-Et-IsTCH were 21.86 +/- 1.77 microM for HeLa cells; 10.37 +/- 1.55 microM for B16 cells; >47 microM for both, nonstimulated and for stimulated, PBMC. Nonstimulated human PBMC appeared to be the most sensitive to the cytostatic IsTCH action; while HeLa cells were the most resistant. N-Et-IsTCH showed more than two or five fold stronger antiproliferative effect toward B16 cells than on HeLa or PBMC cells, respectively, and more than three times intensive activity compared to IsTCH, indicating specificity of N-Et-IsTCH towards inhibition of melanoma cell growth. While increasing concentrations of IsTCH led to decrease in the the PBMC induced suppression of HeLa cell survival; N-Et-Is-TCH in the difference from IsTCH, in dose dependent way contributed to the PBMC induced suppression of HeLa cell survival. In conclusion, the activity of N-Et-Istch on malignant melanoma cells deserves further investigation.

Adenosine contributes to the amine oxidase-mediated spermine cytotoxicity to human myelogenous leukemia K562 cells.

The effects of adenosine, aminophylline, dipyridamole and salbutamol on the amine oxidase-mediated spermine cytotoxicity to KS62 human myelogenous leukemia cells without Ph-chromosome, spontaneously enriched with mildly adherent cells, were studied. In the absence of spermine, adenosine expressed very mild inhibitory action on K562 cell survival, while in combination with the polyamine an almost additive increase in spermine-FBS cytotoxicity was observed. Aminophylline and salbutamol attenuated both spermine-FBS and spermine-FBS-adenosine suppression of cell survival and viability when equimolar concentration of these agents and the adenosine were applied. Pre-treatment of the cells with higher adenosine levels, in the presence of either aminophylline or dipyridamole, or salbutamol, was associated with decreased K562 cell survival, with the appearance of morphological changes in 10-20% of cells. Additional spermine-FBS cytotoxic effect was not observed in cells pre-treated with adenosine-aminophylline, or adenosine-salbutamole, but morphological changes in 10-20% of cells, even in the presence of spermine, was observed again. Dipyridamole alone suppressed very weakly K562 cell survival. In cells pretreated with dipyridamole, in the presence of spermine-FBS, an additive decrease in cell survival was observed. Pre-treatment of cells with dipyridamole and adenosine in presence of spermine-FBS did not result in a decrease of cell survival compared to the one obtained in dipyridamole-spermine-FBS treated cells.

T-2 toxin affects proliferation of three different neoplastic cell lines.

The antiproliferative effect of T-2 toxin (T-2) towards mouse melanoma B16 cells, human myelogenous leukemia K562 cells, and human cervix carcinoma, HeLa cells, was studied. For the first four days of T-2 presence B16 cell survival was decreased in dose dependent fashion. However, cell survival after eleven days T-2 action may be dual: some stimulation of cell growth that was direct function of the number of seeded cells per well was observed and cell survival (for the highest number of seeded cells) six times greater than control, was noticed at 20 nM T-2 toxin concentration. A smaller cell growth stimulation (cell survival more than 3 times higher than control) was observed with a lower cell number seeded per well. Nevertheless, by eleventh day concentrations of T-2 higher than 35 nM completely inhibited B16 cell proliferation. The same trend was noticed for T-2 action towards K562 cells. Treatment of HeLa cells with various T-2 concentrations led to a marked inhibition of cell survival that was more pronounced at the end of 44th or 72nd hour, than after the 20th hour of agent's action. ICs50 values obtained in the present work, suggest that B16 cells were the most sensitive to T-2 antiproliferative action, while HeLa cells were the most resistant. When PBMC were cultured with HeLa cells the antagonism against various T-2 concentrations was observed; cell survival determined after 44, or 72 hours of cells incubation, was less decreased compared to cultures treated with T-2, or with PBMC only. In addition, it was shown that T-2 and cis-DDP had an antagonist effect on HeLa cells survival.

The mechanism of 8-Cl-cAMP action.

8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is a potential new anticancer agent, but its mechanism of action is not clearly defined. In this work we have studied the effect of various heat inactivated and heat untreated human sera in the absence or in the presence of a nonspecific phosphodiesterase (PDE) inhibitor, IBMX, or of nucleoside transport inhibitor and cGMP-specific PDE inhibitor dipiridamole (DP), or of inosine-monophosphatedehydrogenase (IMPDH) inhibitor, tiazofurin, (T), on the antiproliferative 8-Cl-cAMP action towards two human malignant cell lines, K562 and HeLa cells, in vitro. Cell survival was determined 72 hrs after the agents action, using MTT assay. The results obtained, indicated the similar inhibitory effect of 8-Cl-cAMP on HeLa cell survival in the presence of four different heat untreated human sera (IC50 = 4-4.8 microM). Serum heat inactivation caused decrease in 8-Cl-cAMP antiproliferative action depending on the blood donor (IC50 = 23 microM, 15 microM, 19 microM, and 9 microM) and suggesting that some thermolabile ingredient(s) present in sera is involved, at least partially, in the induction or permittance of antiproliferative 8-Cl-cAMP action. K562 Cells were not as much resistant to 8-Cl-cAMP as HeLa cells, or mouse melanoma B16 cells; in the presence of heat untreated FBS, IC50 = 16 microM, while for B16 cells IC50 was 8 microM. Different human sera show different effect on 8-Cl-cAMP action on K562 cells: IC50 = 7.5 microM and 16.5 microM. In the presence of heat inactivated human sera 8-Cl-cAMP IC50 concentrations were higher, with relevant mutual differences. The effect of different sera on 8-Cl-cAMP action was only partly abrogated in the presence of a nonspecific PDE inhibitor, IBMX, suggesting that the serum PDE action is one of the various factors contributing to the induction of 8-Cl-cAMP antiproliferative action. Nucleoside transport inhibitor and cGMP-specific PDE inhibitor dipiridamole inhibited the antiproliferative 8-Cl-cAMP action to HeLa and K562 cells. Tiazofurin and 8-Cl-cAMP acted as antagonists on HeLa, but not on K562 cells.

Protolytic constants of nizatidine, ranitidine and N,N'-dimethyl-2-nitro-1,1-ethenediamine; spectrophotometric and theoretical investigation.

The prototropic exchange equilibria of two drugs, nizatidine (I) and ranitidine(II), and also of structurally related the N,N'-dimethyl-2-nitro-1,1-ethenediamine molecule (III) were investigated. From the changes in electronic spectra in media of various acidity several protonation constants were determined. For pK values were -0.82, 1.95, and 6.67; for ranitidine pK values were 1.95 and 8.13; and for III was 2.60. The hydroxylation equilibrium constant in strongly alkaline media was determined too. Corresponding pK(a) values were 13.23 for I, 13.36 for II and 13.76 for III. Molecular orbital calculations of electronic spectra confirmed that pK 1.95 for I and II, and pK 2.60 for III, are associated with C-protonation of nitroethenediamine fragment, while all pK(a) values correspond to the addition of HO- anion at the same double bond.

To the mechanism of spermine-FBS cytotoxicity toward K562 human myelogenous leukemia cells.

The effects of several compounds acting through adenylate cyclase system and/or influencing prostaglandin biosynthesis on spermine-FBS cytotoxicity to human myelogenous leukemia K562 cells were studied. Salbutamol, a beta 2-adrenoceptor agonist inhibited to a certain extent spermine-FBS cytotoxic action to K562 cells, and propranolol, a beta 2-adrenoceptor antagonist, did not affect this inhibition. Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase, acted suppressing spermine-FBS cytotoxicity to K562 cells. Pretreatment of the cells with dexamethasone did not significantly alter salbutamol-related inhibition of spermine-FBS cytotoxicity. Indomethacin, an inhibitor of cyclooxygenases directly involved in prostaglandin biosynthesis, did not interfere with protective terbutaline effects against spermine-FBS cytotoxicity to K562 cells during the 24-hour period.

Amine oxidase-mediated cytotoxicity of spermine and epinephrine to human myelogenous leukemia K562 cells.

The effects of spermine and beta-adrenoceptor agonists (epinephrine, terbutaline and orciprenaline) in the presence and in the absence of fetal bovine serum (FBS) on human myelogenous leukemia K562 cells viability (V) and survival (N/Nc) were examined. Spermine-FBS significantly decreased both V and N/Nc of K562 cells. Aminoguanidine (AG), an amine oxidase inhibitor, and reduced form of glutathione abolished this effect demonstrating that the spermine-FBS action was amine oxidase-mediated. Epinephrine expressed a strong cytotoxicity to K562 cells which was abolished by pargyline, a specific monoamine oxidase (MAO) inhibitor, as well as by reduced form of glutathione. Terbutaline and orciprenaline exerted no cytotoxic activity to K562 cells cultured in FBS-supplemented medium, independently on the presence of spermine. However, terbutaline at concentrations of over 1 mmol strongly inhibited the cytotoxic effect on spermine-FBS. The relationship between cytotoxicity and chemical structure of beta-adrenoceptor agonists was discussed especially with respect to their stability toward oxidation.

The importance of the specific Z-DNA structure and polyamines in carcinogenesis: fact or fiction.

In this work some aspects of carcinogenesis are given. The importance of the emergence of Z or H DNA structure in the gene, or in the flanking gene sequences for the gene deletion and unusual gene recombination, is discussed. Some considerations on the role of selective pressure (of polyamines, of Mg2+, of the various levels of topoisomerase II, and of ATP) in the process of oncogene amplification, are given too.