Inhibition of functional HER family members increases the sensitivity to docetaxel in human ovarian cancer cell lines.

Human epidermal growth factor (HER) family-targeted therapy combined with standard cytotoxic agents might improve the treatment of ovarian cancer. Human ovarian cancer cell lines OVCAR-3, IGROV-1, and SKOV-3 with differential EGFR, HER2, and HER3 expression levels were used to study whether EGFR-directed (cetuximab) or HER2-directed (trastuzumab, pertuzumab) monoclonal antibodies inhibited cell growth and abrogated activated receptor signaling routes. Possible increase of antiproliferative effects and further activation of caspase-3 as a read-out for apoptosis were analyzed when monoclonal antibodies were combined with docetaxel. Cetuximab alone inhibited cell growth in OVCAR-3 and IGROV-1, which was more pronounced when combined with pertuzumab in OVCAR-3. SKOV-3 cell growth was not significantly affected by any of the antibodies. Cetuximab increased the 50% growth-inhibiting effects of docetaxel in OVCAR-3 and IGROV-1, but not in SKOV-3. Coaddition of pertuzumab to cetuximab plus docetaxel in OVCAR-3 and IGROV-1, and, to a lesser extent trastuzumab in OVCAR-3, inhibited cell growth even further. Caspase-3 activation by docetaxel was enhanced after addition of cetuximab in OVCAR-3 and after addition of cetuximab plus pertuzumab in IGROV-1 and SKOV-3. Functional EGFR-signaling, HER2-signaling, and HER3-signaling routes as shown from abrogation of EGF-stimulated and heregulin-stimulated phosphorylated ERK1/2 by cetuximab, trastuzumab, and pertuzumab, respectively, were shown in OVCAR-3 and IGROV-1, but hardly in SKOV-3. Pertuzumab was able to abrogate phosphorylated HER2 by EGF and heregulin, except in SKOV-3. In conclusion, a combination of docetaxel with inhibitors of HER family members, such as cetuximab plus pertuzumab, may be considered for a clinical trial in ovarian carcinomas with functional receptors.

Interference with actin dynamics is superior to disturbance of microtubule function in the inhibition of human ovarian cancer cell motility.

Cellular movement is mainly orchestrated by the actin and microtubule cytoskeleton in which Rho GTPases closely collaborate. We studied whether cytoskeleton-interfering agents at subtoxic and 50% growth-inhibiting concentrations affect motility of five unselected human ovarian cancer cell lines. Cisplatin and doxorubicin as control cytotoxic agents were not effective, the microtubule-targeting agents docetaxel, epothilone B and vinblastine only marginally inhibited cell motility, while the actin-targeting agent cytochalasin D was most potent in hampering both cell migration and invasion. Disturbance of microtubule dynamics by docetaxel did not importantly affect the cellular structures of beta-tubulin and F-actin. In contrast, hindrance of actin dynamics by cytochalasin D resulted in loss of lamellipodial extensions, induced thick layers of F-actin and disorder in cellular organization. In OVCAR-3 cells the activity of Rac1 was only slightly diminished by docetaxel, but clearly reduced by cytochalasin D. In conclusion, targeting the actin cytoskeleton might provide a means to prevent metastasis formation.

Interaction between celecoxib and docetaxel or cisplatin in human cell lines of ovarian cancer and colon cancer is independent of COX-2 expression levels.

Celecoxib, an inhibitor of cyclooxygenase-2 (COX-2), is being investigated for enhancement of chemotherapy efficacy in cancer clinical trials. We determined whether continuous exposure to celecoxib would increase the antiproliferative effects of a 1-h treatment with docetaxel or cisplatin in four human ovarian cancer cell lines. COX-2 protein could not be detected in these cell lines, because of which three COX-2 positive human colon cancer cell lines were included. Multiple drug effect analysis demonstrated additive to borderline antagonistic effects of celecoxib combined with docetaxel. Combination indices with values of 1.4-2.5 in all cancer cell lines indicated antagonism between celecoxib and cisplatin regardless whether celecoxib preceded cisplatin for 3h, was added simultaneously or immediately after cisplatin. Apoptotic features measured in COX-2-negative H134 ovarian cancer cells and COX-2-positive WiDr colon cancer cells, such as the activation of caspase-3 and the number of cells in sub-G0 of the cell cycle, induced by docetaxel were increased in the presence of celecoxib, but were abrogated upon addition of celecoxib to cisplatin. Moreover, the G2/M accumulation in cisplatin-treated cells was less pronounced when celecoxib was present. Drugs did not affect p-Akt. Celecoxib upregulated p-ERK1/2 in H134 cells, but not in WiDr cells. Platinum-DNA adduct formation measured in WiDr cells, however, was reduced when celecoxib was combined with cisplatin. Taken together, our data demonstrate clear antagonistic effects when celecoxib is given concurrently with cisplatin, which is independent of COX-2 expression levels.

Microtubule-targeting agents inhibit angiogenesis at subtoxic concentrations, a process associated with inhibition of Rac1 and Cdc42 activity and changes in the endothelial cytoskeleton.

Conventional anticancer agents may display antiangiogenic effects, but the underlying mechanism is poorly understood. We determined the antiangiogenic properties of cisplatin, doxorubicin, and the microtubule-targeting agents docetaxel, epothilone B, and vinblastine at concentrations not affecting cell proliferation. We also assessed tubulin and actin morphology and the activity of two key molecules in cell motility, the small Rho GTPases Cdc42 and Rac1. The highest non-toxic concentration (HNTC) of each drug was defined as the concentration inhibiting a maximum of 10% human umbilical vein endothelial cell growth on a 1-hour drug exposure, being for cisplatin 10 micromol/L, doxorubicin 100 nmol/L, docetaxel 10 nmol/L, epothilone B 1 nmol/L, and vinblastine 10 nmol/L. Comparative endothelial cell functional assays using HNTCs for an exposure time of 1 hour indicated that endothelial cell migration in the wound assay, endothelial cell invasion in a transwell invasion system, and endothelial cell formation into tubelike structures on a layer of Matrigel were significantly inhibited by docetaxel, epothilone B, and vinblastine (P < 0.05), but not by cisplatin and doxorubicin. Docetaxel was slightly more efficient in the inhibition of endothelial cell motility than epothilone B and vinblastine. Fluorescence microscopy revealed that only the microtubule-targeting agents affected the integrity of the tubulin and F-actin cytoskeleton, which showed disturbed microtubule structures, less F-actin stress fiber formation, and appearance of nuclear F-actin rings. These observations were associated with early inhibition of Rac1 and Cdc42 activity. In conclusion, HNTCs of microtubule-targeting agents efficiently reduce endothelial cell motility by interference with microtubule dynamics preventing the activation of Rac1/Cdc42 and disorganizing the actin cytoskeleton.