International Journal of Molecular Sciences Article Evaluation for Synergistic Effects by Combinations of Photodynamic Therapy (PDT) with Temoporfin (mTHPC) and Pt(II) Complexes Carboplatin, Cisplatin or Oxaliplatin in a Set of Five Human Cancer Cell Lines Carsten Lange and Patrick J. Bednarski * Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Greifswald, Friedrich-Ludwig-Jahn-Straße 17, 17487 Greifswald, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-3834-420-4883 Received: 24 August 2018; Accepted: 10 October 2018; Published: 16 October 2018 Abstract: The platinum(II) complexes carboplatin (CBDCA), cisplatin (CDDP) and oxaliplatin (1-OHP) are used as anticancer drugs in a large number of tumour chemotherapy regimens. Many attempts have been made to combine Pt(II)-based chemotherapy with alternative treatment strategies. One such alternative anticancer approach is known as photodynamic therapy (PDT), where a non-toxic photosensitizer (PS) produces oxidative stress via the formation of reactive oxygen species (ROS) after local illumination of the affected tissue. A very promising PS is 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC, Temoporfin), which is approved for the treatment of head and neck cancer in Europe. In the present study, a combination of mTHPC-mediated PDT and either CBDCA, CDDP, or 1-OHP was applied to five human cancer cell lines from different tumour origins. Cytotoxicity was determined by the MTT assay and synergistic effects on cytotoxicity were evaluated by calculation of Combination Indices (CI). Synergy was identified in some of the combinations, for example, with 1-OHP in three of the tested cell lines but antagonism was also observed for a number of combinations in certain cell lines. In cases of synergy, elevated ROS levels were observed after combination but apoptosis induction was not necessarily increased compared to a treatment with a single compound. Cell cycle analysis revealed a formation of apoptotic subG1 populations and S phase as well as G2/M phase arrests after combination. In conclusion, pre-treatment with mTHPC-PDT has the potential to sensitize some types of tumour cells towards Pt(II) complexes, in particular 1-OHP but synergy is highly dependent on the type of cancer. Keywords: cancer therapy; Pt(II) complexes; cisplatin; carboplatin; oxaliplatin; photodynamic therapy; mTHPC; combination assay; synergism 1. Introduction The classic cancer therapy with chemotherapeutic agents is, among surgery, immunotherapy, radiation and targeted therapy, one of the central treatment strategies against neoplastic diseases. Platinum(II)-based agents are one group of anticancer drugs in chemotherapy, from which especially cisplatin (cis-diamminedichloridoplatinum(II), CDDP) (1), carboplatin (cis-diammine-1,1-cyclobutanedicarboxylatoplatinum(II), CBDCA) (2) and oxaliplatin (trans-R,R-cyclohexane-1,2-diamineoxalateplatinum(II), 1-OHP) (3) (Scheme1) are used in a very high number of tumour chemotherapies [1–6]. All three mentioned Pt(II) complexes act via the formation of intrastrand and, to a lesser degree, interstrand DNA crosslinks after aquation and Int. J. Mol. Sci. 2018, 19, 3183; doi:10.3390/ijms19103183 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 3183 2 of 21 accompanied activation [4,7,8]. The DNA adducts ultimately lead to the induction of apoptosis [6]. Due to the persistence of severe adverse effects and tumour resistance against the treatment with Pt(II) complexes,Int. many J. Mol. Sci. attempts 2018, 19, x FOR have PEER been REVIEW made to combine Pt(II)-based chemotherapy with2 of 21 alternative treatment strategies, rather than other anticancer drugs [9–11]. One of those alternative anticancer approachesDNA is knownadducts ultimately as photodynamic lead to the induction therapy of (PDT). apoptosis PDT [6]. Due is a to non-invasive the persistence approachof severe for the adverse effects and tumour resistance against the treatment with Pt(II) complexes, many attempts treatmenthave of malignancies, been made to combine where Pt(II)-based a non-toxic chemotherapy photosensitizer with alternative (PS) is treatment activated strategies, by light rather of a certain wavelengththan to generateother anticancer reactive drugs oxygen [9–11]. speciesOne of those (ROS) alternative within theanticancer affected approaches tissue. is The known oxidative as stress then leadsphotodynamic to locally restricted therapy (PDT). cellular PDT death is a non-invasive in the illuminated approach for area. the PDTtreatment displays of malignancies, some advantages over classicwhere cancer a non-toxic therapy, photosensitizer that is, no systemic (PS) is activated effects due by light to non-existent of a certain wavelength toxicity in to the generate absence of light, reactive oxygen species (ROS) within the affected tissue. The oxidative stress then leads to locally selectivityrestricted due to thecellular local death restriction in the illuminated of the illumination area. PDT displays area, some non-invasiveness, advantages over classic no carcinogenicity,cancer no cumulativetherapy, toxicity, that is, no selective systemic retainingeffects due to of non-existent the PS in toxicity tumorous in thetissue absence and of light, effective selectivity recovery due of the healthy surroundingto the local restriction tissue [of12 the,13 illumination]. Disadvantages area, non-invasiveness, include the no restricted carcinogenicity, applicability no cumulative to cancers at advancedtoxicity, stages dueselective to theretaining limited of the penetration PS in tumorous of light tissue into and the effective tumour recovery as well of the as photosensitivityhealthy surrounding tissue [12,13]. Disadvantages include the restricted applicability to cancers at advanced after the treatmentstages due asto thethe limited primary penetration side effect of light due into to athe delayed tumour as clearance well as photosensitivity of the PS. Furthermore, after the it has to be ensuredtreatment that as tumours the primary can side be effect illuminated due to a delayed externally clearance or endoscopically,of the PS. Furthermore, at least it has without to be losing the advantageensured of minimalthat tumours or non-invasivenesscan be illuminated externally [12]. One or endoscopically, very promising at least PS iswithout the reduced losing the porphyrin derivativeadvantage 5,10,15,20-tetra(m-hydroxyphenyl)chlorin of minimal or non-invasiveness [12]. One (mTHPC, very promising Temoporfin) PS is the (reduced4) (Scheme porphyrin1), which was derivative 5,10,15,20-tetra(m-hydroxyphenyl)chlorin® (mTHPC, Temoporfin) (4) (Scheme 1), which approved inwas 2001 approved as Foscan in 2001 asfor Foscan the® palliative for the palliative treatment treatment of head of head and and neck neck cancer [14]. [14 ]. Scheme 1.SchemeChemical 1. Chemical structures structures of the of the Pt(II) Pt(II) complexes complexes cisplatin cisplatin (1),( carboplatin1), carboplatin (2) and (oxaliplatin2) and oxaliplatin (3) (3) as well as theas well photosensitizer as the photosensitizer mTHPC mTHPC (4). (4). EnhancedEnhanced cytotoxic cytotoxic effects effects have have already already been been observed after after the thecombination combination of PDT and of PDT Pt(II) and Pt(II) complexes in some in vitro studies. These included combinations of CDDP and the photosensitizers complexesPhotofrin in some againstin vitro oesophagealstudies. [15] These and includedmouse lymphoma combinations cells [16] or of EtNBS-COOH CDDP and against the photosensitizers small Photofrin againstcell lung oesophagealcancer cells (SCLC) [15] [17]. and CBDCA mouse has lymphoma been combined cells [with16] or9-hydroxypheophorbide EtNBS-COOH against α (9- small cell lung cancerHPbD) cells and (SCLC) radachlorin, [17]. CBDCArespectively, has for been the treatment combined of laryngeal with 9-hydroxypheophorbide cancer cells [18–20] as well αas (9-HPbD) and radachlorin,with verteporfin respectively, against ovarian for the [21] treatment or Photofrin of against laryngeal cervical cancercancer cells cells [22]. [18 1-OHP–20] has as been well as with used in a combined approach with hypericin in colorectal cancer cells [2]. All of these combinations verteporfinled against to enhanced ovarian cytotoxic [21] effects or Photofrin and increased against anticancer cervical efficacy cancer in vitro. cells However, [22]. 1-OHP in all of hasthese been used in a combinedstudies approach just one cancer with cell hypericin line was used in colorectal in the evaluations cancer of cellssynergy. [2]. All of these combinations led to enhanced cytotoxicIn the present effects study, and increasedwe evaluated anticancer the in vitro cytotoxic efficacy andin vitroapoptotic. However, effects of a in combination all of these studies just one cancerof mTHPC-mediated cell line was PDT used and in either the evaluations cisplatin, carboplatin, of synergy. or oxaliplatin on five human cancer cell lines from different tissue origins (bladder, cervix, oesophagus, lung and oral cavity). The insufficient In thetreatment present of study, large solid we evaluatedtumours by PDT the inand vitro persistentcytotoxic severe andadverse apoptotic effects after effects chemotherapy of a combination of mTHPC-mediatedwith Pt(II) complexes PDT and make either the combination cisplatin, of carboplatin, both approaches or interesting oxaliplatin as it onmay five help human overcome cancer cell lines from different tissue