Inhibition of proliferation on some neoplastic cell lines-act of carvedilol and captopril.

The present work examines the effects of beta and alpha1-adrenoceptor antagonist carvedilol, and angiotensin converting enzyme (ACE) inhibitor captopril, on in vitro growth of tumor cell lines derived from breast tumor (MDA-MB-361), melanoma (Fem-x), cervix adenocarcinoma (HeLa) and human myelogenous leukemia (K562). Carvedilol or captopril were applied on malignant cells at 0.1, 1, 5, 10 and 50 micromol. Cell survival was determined 48 hrs after drugs action by MTT. On all cell lines tested, carvedilol was a very potent inhibitor of cell proliferation. The order of sensitivity of various human cell lines to carvedilol's antiproliferative action was: myelogenous leukemia K562 (IC50 = 22.66 +/- 2.14 micromol), > cervix carcinoma HeLa (IC50 = 30.56 +/- 5.16 micromol), > melanoma Fem-x (IC50 = 32.17 +/- 5.75 micromol), > breast tumor MDA-MB-361 (IC50 = 35.04 +/- 2.95 micromol). In contrast, captopril, used in doses from 0.1-50 micromol, was ineffective (IC50 > 50 micromol) to the same cell lines. It is important to note that captopril in concentrations > 1 micromol led to a statistically significant increase in the percent of survived melanoma Fem-x cells (p < 0.05). Understanding the action of these established and clinically accepted agents could provide a basis for design of improved therapeutic regimens in the treatment of cancer diseases.

Cytotoxic and antioxidant activity of Achillea alexandri-regis.

The cytotoxicity and antioxidant properties of herb extracts of Achillea alexandri-regis were studied. Combined chloroform and ethylacetate extracts exhibited a pronounced cytotoxic effect against HeLa cancer cells (IC50 = 25.92 +/- 4.96 microg/ml), and lower cytotoxicity against K562 leukemia cells (IC50 = 48.59 +/- 18.31 microg/ml). The methanol extract was found to be a moderately cytotoxic in vitro agent against HeLa and K562 cells. No suppressive activity was detected on non-malignant peripheral blood mononuclear cells (PBMC). The antioxidant activity of the methanol extract was assessed by DPPH radical scavenging. The methanol extract of A. alexandri-regis showed concentration dependent DPPH radical scavenging activity with IC50 = 36.14 +/- 0.05 microg/ml.

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.

Antimelanoma immunity in vitiligo and melanoma patients.

Cutaneous melanoma and vitiligo are diseases etiology of which evolves around melanocytes. The nature of immunological disturbances associated with these diseases is not elucidated. The experiments performed in this work were aimed to determine antimelanoma immunotoxicity in patients with melanoma and patients with vitiligo. Twelve patients with melanoma, ten patients with vitiligo and seventeen healthy volunteers were studied. The cytotoxicity of PBMC was evaluated indirectly through determination of target melanoma (Fem-x) or control tumor (HeLa) cell survival, in the presence of 15% of AB or autologous sera, by MTT test. The mean values of antimelanoma cytotoxicity in AB serum were similar in both patients groups and in controls. However, the frequency of patients with the enhanced cytotoxicity against melanoma cells, in relation to control tumor cells, was lower in both patients groups than in controls. The intensity of antimelanoma cell-mediated cytotoxicity in melanoma patients, in the presence of autologous serum, was significantly lower in comparison to that found in control subjects and vitiligo patients (p<0.014, in both cases). This indicates that some factors from melanoma patient's sera contribute to impairment of the cytotoxicity of autologous PBMC, while other factors from the serum of vitiligo patients and control subjects enhanced their PBMC antimelanoma cytotoxicity.

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.

Effects of X-ray irradiation on the overexpression of HER-2/Erb-B2 on breast cancer cell lines.

Irradiation is the conventional treatment modality for cancer patients. However, besides its cytotoxic effects on malignant cells it might also affect the biology of surviving cells. Since overexpression of HER-2 receptors on malignant cells is a prerequisite for the therapeutic efficacy of Herceptin, it seems important to know whether previous irradiation changes their overexpression. The experiments performed in this work were aimed to determine whether X-ray irradiation of MDA-MB-361 and MDA-MB-453 breast carcinoma cell lines, besides its cytotoxic action, affects the overexpression of HER-2 protein. Determination of the cytotoxic effect of X-ray irradiation was done using trypan blue test. The breast carcinoma cell responsiveness to herceptin treatment in the presence of 10% fresh human serum (from healthy volunteer's) in the presence or absence of 25 microg/ml of herceptin, in vitro before and after cell-irradiation, was evaluated by MTT test. The degree of HER-2 overexpression was determined by immunocytochemistry, using DAKO HercepTest. Preliminary results obtained in this work showed that X-ray irradiation, besides its cytotoxic effect on malignant cells, could lead to overexpression of HER-2 receptors on (initially by immunocytochemistry, HER-2 negative) tumor cells, indicating change in biology of treated tumor cells. Further investigation in this direction will probably be helpful to elucidate this task in order to improve the selection of irradiated patients for Herceptin therapy.