Aerosol delivery of liposomal formulated paclitaxel and vitamin E analog reduces murine mammary tumor burden and metastases.

Paclitaxel is a chemotherapeutic agent used for the treatment of metastatic breast cancer. 2,5,7,8-Tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid (alpha-TEA) is an analog of vitamin E that inhibits primary tumor growth and the incidence of lymphatic and pulmonary metastases in preclinical animal models. Here, the efficacy of sequential treatment with paclitaxel and alpha-TEA was tested in the BALB/c syngeneic 66cl-4-GFP mammary cancer model. Both agents were formulated into liposomes and delivered by inhalation in an effort to increase anti-tumor efficacy and minimize paclitaxel toxicity. Combination treatment consisting of twelve days of every-other-day treatment with aerosolized paclitaxel (approximately 0.46 microg/mouse/treatment) followed by a daily regimen of aerosolized alpha-TEA (36 microg/mouse/treatment) significantly decreased primary tumor burden when compared to untreated or liposome control groups and was significantly better than individual treatments (P < 0.05). Importantly, combination treatment was significantly better at reducing lung and lymph node micrometastatic foci when compared to control and individual treatment groups (P < 0.05). Immunohistochemical analyses of tumor sections showed combination treatment when compared to liposome control or individual treatments to significantly decrease total number of cells staining positive for the endothelial cell marker CD31 or for the Ki67 marker of cellular proliferation and increase the number of apoptotic (TUNEL positive) tumor cells (P < 0.001). Studies addressing the toxicity of alpha-TEA demonstrated that alpha-TEA formulated in liposomes and delivered by aerosol (72 microg/mouse/day) or gavage (5 mg/mouse/day) for 25 days did not cause blood, liver, or kidney toxicity. In conclusion, sequential inhalation delivery of liposomal-formulated paclitaxel and alpha-TEA produces significantly better anti-tumor outcomes than single treatments.

Vitamin E analog alpha-TEA, methylseleninic acid, and trans-resveratrol in combination synergistically inhibit human breast cancer cell growth.

Alpha-tocopherol ether-linked acetic acid analog [2,5,7,8-tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid (alpha-TEA)] is a novel form of vitamin E effective at killing cancer cells but not normal cells. alpha -TEA alone and together with methylseleninic acid (MSA) and trans-resveratrol (t-RES) were investigated for ability to induce apoptosis, DNA synthesis arrest, and cellular differentiation and inhibit colony formation in human MDA-MB-435-F-L breast cancer cells in culture. The 3 agents alone were effective in inhibiting cell growth by each of the 4 different assays, and 3-way combination treatments synergistically inhibited cell proliferation in each assay in comparison to individual treatments. Furthermore, combinations of alpha -TEA, t-RES, and MSA significantly enhanced levels of apoptosis in human breast (MDA-MB-231, MCF7, and T47D) and prostate (LnCaP, PC-3, and DU-145) cancer cell lines as well as in immortalized but nontumorigenic MCF10A cells but not primary cultures of human mammary epithelial cells. Western immunoblotting confirmed the induction of apoptosis in that the 3 agents induced poly(adenosine diphosphate-ribose) polymerase cleavage, with earlier detection and more complete cleavage seen in the combination treatment. Mechanistic studies showed combination treatments to inhibit cell proliferation via downregulation of cyclin D1 and induce apoptosis via activation of caspases 8 and 9 and downregulation of prosurvival proteins FLIP and survivin. In summary, the combination of alpha-TEA, MSA, and t-RES is more effective than single treatments for inhibiting cell proliferation, inducing cellular differentiation, and inducing cell death by apoptosis in human cancer cells in culture.

Novel vitamin E analogue and 9-nitro-camptothecin administered as liposome aerosols decrease syngeneic mouse mammary tumor burden and inhibit metastasis.

PURPOSE: To test the anticancer properties of a nonhydrolyzable ether-linked acetic acid analogue of vitamin E, 2,5,7,8-tetramethyl-2R-(4R,8R, 12-trimethyltridecyl)chroman-6-yloxyacetic acid (alpha-TEA), and a derivative of camptothecin, 9-nitrocamptothecin (9-NC)singly and in combination against mouse mammary tumor cells (line 66 clone 4 stably transfected with green fluorescent protein; 66cl-4-GFP) cultured in vitro or transplanted subcutaneously into the inguinal region of female BALB/c mice to form established tumors. METHODS: Following in vitro treatment of 66cl-4-GFP cells with alpha-TEA and suboptimal concentrations of 9-NC, singly or in combination, apoptosis was measured by morphological evaluation of nuclei stained with 4',6-diamidino-2-phenylindole (DAPI), and DNA synthesis arrest was measured by tritiated thymidine uptake. For in vivo analyses alpha-TEA and 9-NC, both water-insoluble compounds, were formulated into liposomes using dil-auroylphosphatidylcholine and administered by aerosol to deliver doses calculated to be 36 and 0.4 microg/mouse per day, respectively, (singly or each separately for combined treatments) 7 days per week. RESULTS: Treatment of 66cl-4-GFP cells in culture for 3 days with a combination of alpha-TEA (10 microg/ml; singly produces 38% apoptosis), and suboptimal concentrations of 9-NC(15.6, 31.3, 62.5, or 125 ng/ml; singly produce 2-7% apoptosis), produced 47%, 58%, 64%, and 69% apoptosis. Likewise, combinations of alpha-TEA 9-NC inhibited DNA synthesis more than either agent administered singly. A significant reduction (P< 0.001)in growth of subcutaneous transplanted tumors was observed with liposome-formulated and aerosolized delivery of alpha-TEA + 9-NC to BALB/c mice. The incidence of macroscopic lung metastasis was 83% in control vs 8 % in alpha-TEA-, 9-NC-, or combination-treated mice. Fluorescence microscopic examination of lungs and axillary and brachial lymph nodes showed a statistically significant decrease in metastasis observed in alpha-TEA-,9-NC-, and combination- vs control-treated animals. Analyses of primary tumor tissue for proliferation and apoptosis showed treatment groups to have lower Ki-67 and higher terminal deoxynucleotidyl transferase-mediated nick end labeling, respectively. Treatments showed no measurable effects on two angiogenesis parameters,namely intratumoral blood volume as assessed by hemoglobin content and intratumoral blood vessel density as assessed with CD31 staining. CONCLUSIONS: Combination treatments enhanced antiproliferative and proapoptotic activities in cell culture, and when formulated in liposomes and delivered via aerosolization to treat an aggressive and metastatic syngeneic murine mammary tumor, the combination treatment showed a significant reduction in tumor volume in comparison to either treatment alone. Mechanistically, it appears that neither enhanced apoptosis, reduced cell proliferation,nor reduced blood vessel density can fully account for the enhanced effects of the combination treatment.