Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organellebound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/ photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1 -0.2 mg ml À1 ) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6 -28%). Hypericin (0.1 -0.2 mM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, Po0.05; MCF-7/R: 80.2 and 46.1% increase, Po0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (Po0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy.

Paclitaxel, a natural lipophilic diterpenoid, is currently used in chemotherapy for treatment of some cancers. Paclitaxel acts by blocking the cell cycle at the G1 and M phases and prompts apoptosis. Previously reported studies (Fowler, et al., 2000; McCloskey, et al., 1996) showed that paclitaxel is effective in the treatment of breast cancer and can mediate growth-inhibitory impact of anti-proliferative agents. In our current project we are determining methods to develop and apply 3-dimensional spheroidal cell cultures of the human breast cancer cell line, Hs578T, as a means to investigate the effects of paclitaxel on Hs578T. These methods will be utilized to study, over time, cell viability and morphology in an attempt to more closely mimic in vivo cancer growth under in vitro conditions (Morales & Alpaugh, 2009).(Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and from the West Virginia Higher Education Policy Commission Division of Science and Research SURE Grant Program.)

Although much research has been carried out on the efficacy of possible breast cancer drug treatments in two-dimensional in vitro cell cultures, these may not adequately represent the response of tumors observed in vivo. As an alternative, multicellular three-dimensional spheroids provide an in vitro microenvironment that more closely resembles tumors in vivo. The objective of this study was to develop a 3-dimensional spheroidal cell culture assay using the triple-negative human breast cancer cell line, Hs578T. The results of the project provide a better understanding of the variables of the spheroid culture technique, including cell morphology and viability. It is hoped that future experiments will provide a better understanding of the impact of one anti-proliferative chemotherapy agent, Paclitaxel, on spheroidal Hs578T cells that will, ultimately, provide a model that more closely resembles in vivo cancer growth.