Abstract
The diverse responses of different cancers to treatments such as photodynamic therapy of cancer (PDT) have fueled a growing need for reliable predictive markers for treatment outcome. In the present work we have studied the differential response of two phenotypically and genotypically different breast adenocarcinoma cell lines, MCF7 and MDA-MB-231, to hypericin PDT (HYP-PDT). MDA-MB-231 cells were 70% more sensitive to HYP PDT than MCF7 cells at LD50. MCF7 were found to express a substantially higher level of glutathione peroxidase (GPX4) than MDA-MB-231, while MDA-MB-231 differentially expressed glutathione-S-transferase (GSTP1), mainly used for xenobiotic detoxification. Eighty % reduction of intracellular glutathione (GSH) by buthionine sulfoximine (BSO), largely enhanced the sensitivity of the GSTP1 expressing MDA-MB-231 cells to HYP-PDT, but not in MCF7 cells. Further inhibition of the GSH reduction however by carmustine (BCNU) resulted in an enhanced sensitivity of MCF7 to HYP-PDT. HYP loading studies suggested that HYP can be a substrate of GSTP for GSH conjugation as BSO enhanced the cellular HYP accumulation by 20% in MDA-MB-231 cells, but not in MCF7 cells. Studies in solutions showed that L-cysteine can bind the GSTP substrate CDNB in the absence of GSTP. This means that the GSTP-lacking MCF7 may use L-cysteine for xenobiotic detoxification, especially during GSH synthesis inhibition, which leads to L-cysteine build-up. This was confirmed by the lowered accumulation of HYP in both cell lines in the presence of BSO and the L-cysteine source NAC. NAC reduced the sensitivity of MCF7, but not MDA-MB-231, cells to HYP PDT which is in accordance with the antioxidant effects of L-cysteine and its potential as a GSTP substrate. As a conclusion we have herein shown that the different GSH based cell defense mechanisms can be utilized as predictive markers for the outcome of PDT and as a guide for selecting optimal combination strategies.
Highlights
Photodynamic Therapy is a photomedical treatment using a photosensitive substance, photosensitizer (PS), which upon irradiation by light at the appropriate wavelength, interacts with biomolecules or molecular oxygen to produce reactive species
Chemicals and reagents RPMI 1640 without phenol red, L-Glutamine, penicillin/streptomycin, trypsin, dimethylsulfoxide (DMSO), Antimycin A (ANTI-A), N-Acetyl-L-cysteine (NAC), Ethylenediaminetetraacetic acid (EDTA), trizma® hydrochloride (TRIS-HCL), trizma® base (TRIS-BASE), polyethylene glycol sorbitan monolaurate (TWEEN 20), bovine serum albumin (BSA), Buthionine sulfoximine (BSO), Carmustine (BCNU), Triton X-100, thiazolyl blue tetrazolium bromide (MTT), β-Nicotinamide adenine dinucleotide reduced disodium salt (NADH), metaphosphoric acid (MPA), triethanolamnine (TEAM), anti-γ-tubulin, Glutathione S-Transferase (GST) Assay Kit and sodium pyruvate were purchased from Sigma-Aldrich Norway AS (Oslo, Norway), Anti-glutathione peroxidase 4 (GPX-4) (H-90) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.) and anti-glutathione S transferase p1 (GSTP1) (3F2) from Cell Signalling Technology, Inc. (Danvers, MA, U.S.A.)
In a course of photodynamic therapy of cancer (PDT) experiments with HYP on MCF7 and MDA-MB231 cells, we found that MCF7 cells were more resilient to PDT than MDA-MB-231 cells (Fig. 1)
Summary
Photodynamic Therapy is a photomedical treatment using a photosensitive substance, photosensitizer (PS), which upon irradiation by light at the appropriate wavelength, interacts with biomolecules or molecular oxygen to produce reactive species. In recent years molecular biology-based research, including deep sequencing analyses, has documented the large tumor heterogeneity even between tumors of the same origin and sub-classification [1] This has led to development of personalized medicine based on predictive markers for treatment response and is expected to result in the use of approved drugs and treatment modalities on only a subfraction of tumors today treated uniformly [2]. Standard therapies include ionizing radiation, various chemotherapeutic and hormone based therapies as well as mAbs such as Herceptin These treatment modalities induce resistance mechanisms that could influence second line treatments such as PDT. In that study MCF7 cells were practically insensitive to TPCS2a PDT, while MDA-MB-231 were very efficiently decimated by the same treatment
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