metastat and Breast-Neoplasms

Paclitaxel, a chemotherapeutic agent used in the treatment of breast cancer and other solid tumor types, including ovarian and lung, causes a dose‑dependent neuropathic pain, which limits its use. Chemically modified tetracycline‑3 (COL‑3) has anticancer properties and was previously reported to inhibit neuroinflammation and protect against paclitaxel‑induced neuropathic pain (PINP) in mice models. However, it is not known whether it affects the anticancer activities of paclitaxel. Thus, the aim of the present study was to evaluate the effect of COL‑3 on the anticancer activity of paclitaxel on the breast cancer cell lines MCF‑7 (estrogen receptor‑positive), pII [estrogen receptor‑negative (ER‑ve)] and MDA‑MB‑231 (ER‑ve). Cell proliferation, apoptosis and cell cycle stage were determined using an MTT assay, Annexin V/7‑aminoactinomycin D and flow cytometry. The expression of various signaling molecules was determined with ELISA‑based proteome profiling and western blotting. Additionally, the degree of cell invasion was determined with a Matrigel assay and caspase‑3 activity was determined with a colorimetric assay. Treatment with paclitaxel or COL‑3 alone inhibited cell proliferation in a concentration‑dependent manner in all cell lines. The anti‑proliferative effects of paclitaxel and COL‑3 in combination varied from synergism against MDA‑MB‑231 and pII cells to notably additive and slight antagonism against MCF‑7 cells. In the highly proliferative and invasive pII cells, the observed synergistic anti‑proliferative effect was partially through the induction of apoptosis via modulation of caspase‑3 levels and activity, and P70S6K phosphorylation, but not cell cycle arrest. COL‑3 inhibited the invasion of pII cells in a concentration‑dependent manner partially through inhibiting total matrix metalloproteinase activity. The combination regimen significantly inhibited the expression of two proteases, ADAM metallopeptidase with thrombospondin type 1 motif 1 and proteinase 3. In conclusion, the combination of paclitaxel and COL‑3 indicated additive to synergistic anti‑proliferative effects on breast cancer cells mediated partially via the induction of apoptosis. The combination regimen could further inhibit invasion and metastasis. Thus, COL‑3 could be a beneficial adjunct to a paclitaxel‑based anticancer regimen to improve therapeutic outcome and reduce the adverse effects of paclitaxel, primarily PINP.

Topics: Apoptosis; Breast Neoplasms; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Female; Humans; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Paclitaxel; Tetracyclines

Inhibition of tumour cell proliferation, invasion and metastasis by chemically modified tetracyclines has been ascribed to inhibition of matrix metalloproteinase (MMP) activity.. Exposure of the human breast carcinoma cell line MDA-MB-231 or its MMP-9-overproducing transfected clone (E-10) to 6-demethyl, 6-deoxy, 4-de [dimethylamino]-tetracycline (CMT-3), a chemically modified non-antimicrobial tetracycline followed by analysis using gelatinase activity assay, zymography, degradation of radiolabelled extracellular matrix (ECM), Western blotting, TNF-alpha ELISA and cell viability assays.. CMT-3 treatment results in diminution in extracellular MMP-9 protein levels as well as inhibition of gelatinase activity. This prevents cell-mediated ECM degradation without inducing general cytostasis or cytotoxicity. Culturing E-10 cells in 10 or 20 microM CMT-3 diminished secreted MMP-9 levels by 45% or 60%, respectively, but did not affect levels of most other secreted proteins, including tissue inhibitor of Metalloproteinases (TIMP-1). ECM degradation by E-10 cells or their conditioned medium was inhibited by approximately 20%-30% in the presence of 20 microM CMT-3, reflecting inhibition of MMP-9 activity in addition to diminution of released MMP-9 levels. TNF-alpha levels were also diminished in E-10 conditioned medium in the presence of CMT-3, but cell viability, measured by MTS reduction and cytosolic LDH retention, was unaffected.. It is proposed that the reduction in ECM-degradative activity reflects diminished levels of expression as well as inhibition of enzymatic activity of MMPs released by cells in the presence of CMT-3. These multiple effects of CMT-3 may offer promise for use in suppressing tumour invasion, and if used in conjunction with other chemotherapy agents, may lead to more successful treatment of cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Survival; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Gelatinases; Humans; Immunoblotting; Matrix Metalloproteinase 9; Tetracyclines; Tissue Inhibitor of Metalloproteinase-1