Abstract. The purpose of this study was to evaluate the cytotoxic effect of thalidomide on 4T1 and 4THMpc mouse breast cancer cell lines. Mouse breast cancer cells (4T1) and cells derived from metastatic lesions (4THMpc) were treated with various doses of thalidomide [10 -2 -100 µM dissolved in dimethyl sulfoxide (DMSO) as recommended] and 1.4 µM DMSO (maximum DMSO concentration in the highest thalidomide dose) as a DMSO control against the untreated control groups. MTT was used to evaluate the cytotoxic effects of the treatments. Therefore, we investigated the role of thalidomide on apoptosis. A fluorometric EnzChek caspase-3 enzyme activity assay kit was used to evaluate the apoptotic effects of thalidomide. Thalidomide dissolved in DMSO exhibited cytotoxic effects on 4T1 and 4THMpc cells compared to the control groups incubated without any supplement. Treatment with thalidomide resulted in apoptosis of mouse breast cancer cells in a time-and dose-dependent manner as demonstrated by caspase-3 enzyme activity. However, DMSO alone suppressed cell proliferation more effectively than thalidomide. In cultured mouse breast cancer cells the inhibitory effect of thalidomide may be partially attributed to the solvent DMSO alone. IntroductionBreast cancer, which is the second leading cause of cancer mortality in women, occurs at a high frequency (1). Although there have been significant advances in diagnosing and treating breast cancer, a number of major unresolved clinical and scientific problems remain and investigations are ongoing (2).Thalidomide [a(N-phthalimido)-glutarimide, a derivative of glutamic acid with two rings and two optically active forms] is an odorless, white crystalline compound with low solubility in water and has a cytotoxic effect on a number of cancer cell lines, including breast cancer (3,4).Thalidomide has been shown to be clinically useful in a number of situations due to its ability to inhibit TNF-α synthesis. However, its use is restricted by potentially serious side effects, including teratogenicity (5). Furthermore, insolubility may cause serious problems in terms of systemic bioavailability. The major solvent of thalidomide is dimethyl sulfoxide (DMSO) and it has been shown to mimic the cytotoxic effects of thalidomide (6).The aim of this study was to evaluate the effect of thalidomide and DMSO on the viability of cultured 4T1 and 4THMpc mouse breast cancer cell lines. Materials and methodsThalidomide. Thalidomide was purchased from SigmaAldrich (Cat. No. T 144; St. Louis, MO, USA). A 100 µM stock solution dissolved in DMSO (14.08 mM) was prepared and then the solution was aliquoted into standard Eppendorf tubes in quantities of 500 µl for daily assays. These aliquots were stored at -70˚C until required.Cell lines and in vitro cell culture conditions. 4T1 breast cancer cells and 4T1 heart metastases post capsaicin (4THM), a cell line obtained from orthotopically transplanted 4T1 breast cancer cells, were used in this study. The cell lines were a kind gift from Dr Nuray Erin (Akdeniz Unive...
Abstract. thalidomide is an anti-angiogenic agent that is used in the treatment of cancer. however, in many cases, particularly in patients with breast cancer, thalidomide treatment alone is insufficient and must be combined with other drugs or therapies. in the clinical setting, thalidomide is most commonly used in combination with radiation therapy. however, the exact mechanisms of its effect are unkown. radiotherapy alters the expression of substance p, which is considered a crucial pro-angiogenic peptide. to determine whether thalidomide and radiotherapy in combination overcome the limitations of each as monotherapy, we examined the effects of the combination on the growth of breast cancer cells as well as on the expression of substance p in vitro. mouse breast cancer cells (4t1) and cells produced from metastatic lesions (4thmpc) were treated with radiotherapy (rt) (45 Gy) alone, thalidomide (thal) (40 µg/ml) alone or combination therapy (40 µg/ml thal + 45 Gy rt), and compared with control cells. mtS, live/dead and trypan blue exclusion assays were used to evaluate the cytotoxic effects of the treatments. the levels of substance p in the conditioned media and in the cell lysates were determined by a substance p EliSa kit, and changes in the protein content were analyzed by Western blotting. Thalidomide alone resulted in a significant inhibition in the growth of the 4t1 (34.1%) and 4thmpc (52.6%) cell lines. rt alone inhibited the growth of the 4t1 (19.2%) and 4thmpc (23.31%) cell lines. the combination therapy enhanced the growth inhibition noted in the 4t1 (47.9%) and 4thmpc (62.03%) cell lines. the expression of substance p in the conditioned media and in the cell lysates increased within 72 h of RT. This increase was significantly enhanced with the combination therapy. these data indicate that thalidomide inhibits breast cancer cell growth and potentiates the anti-tumor effects of radiation at appropriate doses.
Abstract. Radiotherapy is widely used in the treatment of cancer. On the other hand, endostatin is considered to be a potent inhibitor of angiogenesis. Therefore, to test whether ES combined with RT overcomes the limitations of each monotherapy the present study investigated the effects of endostatin (ES), radiotherapy (RT) or combination therapy on the growth of mouse breast cancer cells as well as on the expression of substance P in vitro. The breast cancer cell lines 4T1 and 4THMpc were treated with recombinant murine ES (0.5, 1, 2, 4 and 8 µg/ml) alone, RT (45 Gy) alone or as a combination therapy. Cell proliferation was evaluated using an MTS assay and the results were verified by the Live/Dead assay. Immunoprecipitation and Western blotting analysis were performed to determine whether the substance P levels of the two cell lines occurred due to substance P content. Results showed that ES alone resulted in a low but significant inhibition in the growth of 4T1 and 4THMpc cell lines (27.63 and 21.75%, respectively). RT alone inhibited the growth of 4T1 (30.81%) and 4THMpc (39.64%) cells. A combination of ES with RT enhanced growth inhibition in the cells (83% in 4T1 and 80% in 4THMpc). The amount of substance P, both in the conditioned media and the cell lysates, increased within 72 h after RT. This increase was inhibited when ES and RT were applied in combination. These data indicate that ES inhibits the in vitro growth of breast cancer cells and potentiates the antitumor effects of RT at appropriate doses via alteration of the amount of substance P and thus an increase of radioresponse.
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