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Proadifen (SKF-525A) is a P450 monooxygenase inhibitor with potential anti-proliferative activity and the ability to potentiate the toxicity of hypericin-mediated photodynamic therapy and mitoxantrone via alteration of ABC transport proteins. Elevated expression of some ABC transporters may also determine the efficacy of cisplatin-based chemotherapy. Thus, the purpose of this study was to investigate the ability of proadifen to sensitize A2780 and A2780cis ovarian cancer cells to cisplatin (CDDP). Herein, we show for the first time that proadifen sensitized resistant ovarian cancer cells to CDDP-induced cell death. The chemosensitizing effect of proadifen on CDDP action was also confirmed by MTT assays in multicellular spheroids. The possible mechanisms responsible for the enhanced cytotoxicity of proadifen/CDDP combined treatment may be attributed to a decrease of reduced relative glutathione levels, downregulation of multidrug resistance-associated proteins 1 and 2 (MRP1, MRP2) and attenuation of survivin expression. Taken together, our results indicate that proadifen is a promising compound for further in vivo experiments related to overcoming multidrug resistance and sensitization of resistant ovarian carcinoma to CDDP.

HL-60 cancer cells were treated with a series of novel acridine derivatives (derivatives 1-4) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. Binding studies of derivatives 1-4 with calf thymus DNA were also performed using a number of techniques (UV-Vis and fluorescence spectroscopy, thermal denaturation, linear dichroism and viscometry) to determine the nature of the interaction between the compounds and ctDNA. The binding constants for the complexes of the studied acridine derivatives with DNA were calculated from UV-Vis spectroscopic titrations (K=3.1×10(4)-2.0×10(3)M(-1)). Some of the compounds showed a strong inhibitory effect against Topo II at the relatively low concentration of 5μM. Topo I/II inhibition mode assays were also performed and verified that the novel compounds are topoisomerase suppressors rather than poisons. The biological activities of derivatives were studied using MTT assay and flow cytometric methods (detection of mitochondrial membrane potential, measurement of cell viability) after 24 and 48h incubation. The ability of derivatives to impair cell proliferation was tested by an analysis of cell cycle distribution.

Background Hypericin-mediated photodynamic therapy (HY-PDT) has recently captured increased attention as an alternative minimally invasive anticancer treatment, although cancer cells may acquire resistance. Therefore, combination treatments may be necessary to enhance HY-PDT efficacy. Histone deacetylase inhibitors (HDACis) are often used in combination treatments due to their non-genotoxic properties and epigenetic potential to sensitize cells to external stimuli. Therefore, this study attempts for the first time to investigate the therapeutic effects of HDACis in combination with visible light-mediated PDT against cancer. Specifically, the colorectal cancer cell model was used due to its known resistance to HY-PDT. Results Two chemical groups of HDACis were tested in combination with HY-PDT: the hydroxamic acids Saha and Trichostatin A, and the short-chain fatty acids valproic acid and sodium phenylbutyrate (NaPB), as inhibitors of all-class versus nuclear HDACs, respectively. The selected HDACis manifest a favorable clinical toxicity profile and showed similar potencies and mechanisms in intragroup comparisons but different biological effects in intergroup analyses. HDACi combination with HY-PDT significantly attenuated cancer cell resistance to treatment and caused the two HDACi groups to become similarly potent. However, the short-chain fatty acids, in combination with HY-PDT, showed increased selectivity towards inhibition of HDACs versus other key epigenetic enzymes, and NaPB induced the strongest expression of the otherwise silenced tumor suppressor CDKN1A, a hallmark gene for HDACi-mediated chromatin modulation. Epigenetic regulation of CDKN1A by NaPB was associated with histone acetylation at enhancer and promoter elements rather than histone or DNA methylation at those or other regulatory regions of this gene. Moreover, NaPB, compared to the other HDACis, caused milder effects on global histone acetylation, suggesting a more specific effect on CDKN1A chromatin architecture relative to global chromatin structure. The mechanism of NaPB + HY-PDT was P53-dependent and likely driven by the HY-PDT rather than the NaPB constituent. Conclusions Our results show that HDACis potentiate the antitumor efficacy of HY-PDT in colorectal cancer cells, overcoming their resistance to this drug and epigenetically reactivating the expression of CDKN1A. Besides their therapeutic potential, hypericin and these HDACis are non-genotoxic constituents of dietary agents, hence, represent interesting targets for investigating mechanisms of dietary-based cancer prevention.

Since many studies have suggested the impact of dietary polyunsaturated fatty acids on cancer progression and prognosis, there is an assumption of possible pre-sensitizing effects of their application in combined treatment. The present work evaluates modulation of photodynamic therapy (PDT) with hypericin by pre-treatment with n-3 and n-6 fatty acids in HT-29 and HeLa tumour cells. We observed stimulation of cytotoxic effects by docosahexaenoic acid (n-3) and arachidonic acid (n-6) in several stages of action in both cell lines. Treatment with either fatty acids or photodynamic therapy alone induced apoptosis in a dose- and time-dependent manner; however the effect was even more striking in mutual combination applied as pre-treatment with fatty acids prior to photodynamic therapy. Moreover, the combination also induced changes in membrane lipid composition leading to alteration in cell membrane fluidity. Increased toxicity of combined treatment was also confirmed by the presence of oxidative stress demonstrated by ROS production, RNS accumulation and increased presence of lipoperoxides. In conclusion, we suggest that pre-treatment with polyunsaturated fatty acids may contribute to cytotoxic effects induced by photodynamic therapy with hypericin.

Multidrug resistance caused by the overexpression of ABC transporter proteins in cancer cells remains a major obstacle limiting chemotherapy efficacy. Drugs inhibiting these transporters have been shown to increase the anti-proliferative properties of chemotherapeutics. As we previously described, proadifen, a P450 monooxygenase inhibitor, might also be able to inhibit some ABC transporters, including breast cancer resistance protein (BCRP). Because mitoxantrone (MTX) is a strong BCRP substrate and is often used in the treatment of leukemia, we investigated the effect of 24 h proadifen pre-treatment on the cytotoxicity of MTX in leukemic cell lines that are sensitive to MTX (HL-60) and MTX-resistant ABCG2-overexpressing subclone (cBCRP). We show for the first time that proadifen is able to enhance the cytotoxic properties of MTX in cBCRP cells, particularly through the inhibition of BCRP expression and activity. This proadifen-MTX synergism was also mediated by the inhibition of various cellular proteins engaged in apoptosis, including Mc-1, Bcl-xL, survivin and activation of procaspase-3. Proadifen also decreased the expression of γH2AX, which is involved in the recruitment of reparation proteins. Moreover, the inhibition of DNA damage repair proteins Ku86 and B23 after proadifen treatment indicate a possible role of proadifen in DNA repair blockage, thus suppressing the reparation rate of MTX-induced DSBs.

Summary Background Pretreatment with 5-LOX pathway inhibitor MK-886 potentiates cytotoxic effects of photodynamic therapy mediated by natural photosensitizer, hypericin. In this study, we focused on elucidating mechanisms beyond the increased efficacy of combined treatment. Methods Metabolic activity/viability, caspase-3 activation/mitochondrial membrane potential dissipation, intracellular hypericin level, glutathione level and redox status (NAD(P)H/oxidized flavins ratio) analyses, as well as drug efflux assays, were performed by flow cytometry. Changes in protein expression of ATP-binding cassette transporters, GDF-15 and other selected proteins were evaluated by Western blotting. Silencing of gdf-15 was carried out to verify its role in response to treatment. Results MK-886 pretreatment led to a concentration-dependent increase in intracellular hypericin content, accompanied by changes in ATP-binding cassette transporters levels and efflux efficiency. Intracellular accumulation of cytokine GDF-15 correlated with increased cell death markers; however, the impact of gdf-15 silencing on the evaluated markers was negligible. A marked decrease in the glutathione level of a majority of cells was observed after more toxic combination treatment. Conclusion The significant increase in cell death markers after combination treatment confirms the potentiating effect of MK-886 on hypericin-mediated photodynamic therapy in HT-29 and MCF-7 cells. Although BCRP downregulation was not confirmed as leading mechanism responsible for elevated levels of hypericin content, changes in expression and efflux activity of ABC transporters caused by MK-886 suggest its potential in combination treatment with drugs that are substrates of these transporters, predominantly MRP1. However, complex cellular response to MK-886 pretreatment needs to be considered and further elucidated.

St. John's wort (SJW, Hypericum perforatum L.) is a commonly used natural antidepressant responsible for the altered toxicity of some anticancer agents. These interactions have been primarily attributed to the hyperforin-mediated induction of some pharmacokinetic mechanisms. However, as previously demonstrated by our group, hypericin induces the expression of two ABC transporters: multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). Because cisplatin (CDDP) and mitoxantrone (MTX) are potential substrates of ABC transporters, we investigated the effect of 24h hypericin pre-treatment on the cytotoxicity of CDDP and MTX in human cancer cell lines. CDDP-sensitive and -resistant ovarian adenocarcinoma cell lines A2780/A2780cis, together with HL-60 promyelocytic leukemia cells and ABCG2-over-expressing cBCRP subclone, were used in our experiments. We present CDDP cytotoxicity attenuated by hypericin pre-treatment in both A2780 and A2780cis cells and MTX cytotoxicity in HL-60 cells. In contrast, hypericin potentiated MTX-induced death in cBCRP cells. Interestingly, hypericin did not restore cell proliferation in rescued cells. Nevertheless, hypericin did increase the expression of MRP1 transporter in A2780 and A2780cis cells indicating the impact of hypericin on certain resistance mechanisms. Additionally, our results indicate that hypericin may be the potential substrate of BCRP transporter. In conclusion, for the first time, we report the ability of hypericin to affect the onset and/or progress of CDDP- and MTX-induced cell death, despite strong cell cycle arrest. Thus, hypericin represents another SJW metabolite that might be able to affect the effectiveness of anti-cancer drugs and that could interact with ABC transporters, particularly with BCRP.

Summary Background Photodynamic therapy (PDT) is a highly efficient approach for tumour therapy, though it also has its drawbacks, too. There are multiple mechanisms involved in cell death regulation that can be successfully targeted for improvement of PDT in particular cases. We assumed, however, that the potential to manage radical stress might be the primary factor responsible for resistance to hypericin-mediated PDT (HY-PDT). Methods We compared the sensitivity of six colon-derived cancer cell lines to HY-PDT at IC50 equitoxic doses acquired by formazan-based (MTT) assay. Intracellular hypericin content, cell survival/metabolic activity, caspase-3 activation/mitochondrial membrane potential dissipation, apoptosis, glutathione level, redox status (NAD(P)H/oxidized flavins ratio) and Western blot analyses of proteins relevant in apoptosis regulation were measured to demonstrate differences between tested cell lines. Results Analyses revealed a whole spectrum of responses from insignificant to high cytotoxicity, despite the MTT-based “equitoxicity”. Further critical evaluation of multiple parameters linked to cell physiology and proteomics proved that intracellular hypericin content, glutathione level or redox status demonstrate partial but not direct correlation with resistance to HY-PDT, when considered separately. However, their logical conjunction did copy the trend of cellular resistance. Conclusions We may conclude that intracellular level of hypericin and glutathione together with redox state of the target cell represent a potential combination of parameters responsible for the primary cytotoxicity of HY-PDT. We also present evidence that cytotoxic assays, such as the MTT, should be accompanied with other tests of cell survival/cytotoxicity in order to avoid incorrect conclusions.

Preferential uptake of photosensitizer by tumour tissue is an elementary prerequisite of effective and successful photodynamic therapy (PDT). Therefore intracellular concentration of photosensitizer is one of the limiting factors affecting PDT efficiency. Hypericin (HY) has found applications in photodynamic diagnostics solely due to its high specificity for tumour cells and tissues. However, here we suggest that not only HY uptake, but importantly also the cell ability to manage oxidative stress induced by HY-PDT can be important decisive factors finally affecting the cell death response. We showed that despite the higher accumulation of HY in FHC human fetal colon epithelial cells compared to HT-29 colon adenocarcinoma cells, the cytotoxic effects of this photosensitizer were more pronounced in the latter cell line, and this was associated with enhanced accumulation of HY-PDT-induced reactive oxygen species (ROS).