The effects of Ce6-mediated sono-photodynamic therapy on cell migration,apoptosis and autophagy in mouse mammary 4T1 cell line

Purpose

Sono-Photodynamic therapy (SPDT) is an alternative therapy which claims to enhance the anti-cancer effects by combining sonodynamic therapy (SDT) with photodynamic therapy (PDT). In the present study, we investigated the effects of chlorin e6 (Ce6) mediated SPDT on migration, apoptosis and autophagy in mouse mammary 4T1 cancer cells, and its underlying mechanisms.

Materials

Cell migration was determined by wound healing assay. Apoptosis was analyzed using annexin V-PE/7-ADD staining as well as Hoechst 33342 staining. Changes of mitochondria membrane potential (MMP) was evaluated by flow cytometry. Formation of acidic vesicular organelles (AVOs) during autophagy was observed with fluorescence microscope by MDC staining. Immunofluorescence assays were performed to detect the co-localization of LC3 and Lamp2. Western blotting was employed to analyze the activity of the apoptosis related proteins Caspase-3, PARP, Bax and Bcl-2, as well as the autophagy associated processing of LC3-I to LC3-II and Beclin-1 expression.

Results

Ce6 mediated SPDT further enhanced cell migration inhibition, significantly triggered cell apoptosis, nuclear condensation and MMP drop. Cleaved Caspase-3 and PARP increased dramatically after Ce6-SPDT, accompanied by decreased Bcl-2 expression, while the expression of Bax remained stable. Additionally, AVOs formation, co-localization of LC3 and Lamp2 occurred following Ce6-SPDT and simultaneously accompanied by LC3-II processing and increased Beclin-1 expression.

Conclusions

Ce6-SPDT could enhance cell migration inhibition, and induce mitochondria-dependent apoptosis as well as autophagy in 4T1 cells.     

新型二氢卟吩类光敏剂的设计及理论计算研究

本文采用密度泛函理论（DFT）和含时密度泛函理论（TD-DFT）方法对一系列具有π共轭结构的二氢卟吩类光敏剂进行了理论模拟研究,计算了此类衍生物的吸收光谱特征、分子内电荷分布特点以及三重激发态特性。结果显示,在二氢卟吩大环结构中共轭引入给电子芳香基团（对甲氧基苯基）,可使化合物（B2）的Q带吸收发生红移,分子内负电荷聚集增强,减小单重态-三重态能级差（ΔEST）,有利于发生系间窜越,使单线态氧产量增加,导致强效的肿瘤生长抑制。并且在B2分子大环中心配合Pd原子会进一步减小ΔEST。因此,对于二氢卟吩类光敏剂,引入给电子共轭基团并配合金属原子是提高其PDT治疗效果的有效改进策略。     

Cyclophane-Type Chlorin Dimers from Dynamic Covalent Chemistry of 2,18-Porphyrinyl Dicyanomethyl Diradicals

2,18-Bis(dicyanomethyl)-substituted Ni^II porphyrin 8 and Zn^II porphyrin 11 were prepared and subjected to oxidation with PbO₂ in CH₂Cl₂ at 298 K to give cyclophane-type chlorin dimers ( 9 )₂ and ( 12 )₂ as a consequence of double recombination of biradicals 9 and 12 , respectively. Dimer ( 9 )₂ takes a syn-conformation of two distorted Ni^II chlorins but ( 12 )₂ takes an anti-conformation of relatively planar Zn^II chlorins. At 298 K, dimer ( 9 )₂ is stable and its ¹H NMR spectrum is sharp but becomes broad at high temperature, while the ¹H NMR spectrum of ( 12 )₂ is considerably broad even at 298 K but becomes sharper at low temperature. These results indicate that the chlorin dimers dissociate to radical species, but the activation barrier of the dissociation of ( 12 )₂ is much less than that of ( 9 )₂. The involvement of diradicals in dynamic covalent chemistry has been suggested by thermal scrambling of hetero dimer ( 16 )₂ to give homo dimers ( 9 )₂ and ( 15 )₂.     

Fluorinated porphyrinoids and their biomedical applications

Porphyrins, phthalocyanines, chlorins and corroles create a compact group of macrocyclic compounds of established utility in medicine and technology. Fluorine atom insertion to their structures belongs to one of many ongoing approaches for improving their potency. The presence of fluorine in the structure of a photosensitizer may enrich it with required pharmacokinetic features. Photostability, high level of singlet oxygen production, lipophilicity and selective accumulation in tumor cells have made the fluorinated porphyrinoids potential entities for photodynamic therapy. Moreover, photosensitizers possessing intrinsic fluorescence may be applied as agents in photodynamic diagnosis of cancer. Noteworthy, magnetic resonance of fluorinated compounds constitutes excellent probe for sensitive and minimally invasive imaging.Porphyrins constitute the largest group within fluorinated porphyrinoids of potential anticancer and antimicrobial properties. Skillful combination of fluorine substituents with other functional groups in their structures, including glycol or sugars led to novel molecules possessing outstanding phototoxicity in both in vitro and in vivo studies. Furthermore, fluorinated porphyrins were researched as biomimetic systems imitating the active sites of some enzymes. Fluorinated chlorins are considered as promising photosensitizers due to a strong absorption and the least harmful effects to human tissues. Fluorine containing groups introduced to the periphery of phthalocyanines improved their solubility in common solvents and thus enhanced applications in in vitro and in vivo researches. Lately investigated porphyrinoids belonging to corroles showed on one hand potential as photosensitizers, but on the other hand they were found as catalytic anti-oxidants for attenuation of diabetes mellitus.     

Square‐Wave Voltammetry of Sodium Copper Chlorophyllin on Glassy‐Carbon and Paraffin‐Impregnated Graphite Electrode

Electrochemical oxidation of sodium copper chlorophyllin (CHL) has been investigated at a glassy‐carbon (GC) and paraffin‐impregnated graphite electrode (PIGE) using square‐wave voltammetry (SWV). Square‐wave voltammograms of other two chlorin‐type compounds, namely chlorin e₆ and chlorophyll a, have been studied as well. The measurements were performed in the pH range between 7 and 11. The square‐wave frequency was changed between 8 and 1000 Hz. The oxidation of studied chlorins is a complex, pH‐independent, reversible or quasireversible process, followed by the chemical transformation of the product. The product of the EC reaction of CHL is an electroactive π? π dimer, which strongly adsorbs on the electrode surface and undergoes further oxidation at more positive potential. The electrooxidation of the adsorbed dimer is a pH‐independent irreversible process with the formation of an electroinactive film. The voltammetric behaviour of chlorin e₆ on PIGE was qualitatively similar to that of CHL. The SW voltammograms of chlorin e₆ recorded on GCE and of chlorophyll a recorded on PIGE consisted of only one peak. The SW responses of studied compounds strongly depend on the stabilization of the reaction intermediate by adsorption to the electrode surface.     

[2-Methylazeteochlorinato]Ni(II): a pyrrole ring-contracted chlorin analogue

The formal replacement of one of the pyrrole rings in [meso-tetraphenylporphyrinato]Ni(II) (5Ni) by an azete moiety is reported. Thus, reaction of known chlorophin monoaldehyde 7Ni (made in three steps from 5Ni) with methyl-Grignard, followed by an acid-catalyzed ring-closure reaction, generates the title compound [meso-tetraphenyl-2-methylazeteochlorinato]Ni(II) (10Ni) in a rational and scalable process in good yields. The UV-vis spectroscopic properties of this chromophore are, as expected for this chlorin analogue, red-shifted when compared to the corresponding [porphyrinato]Ni(II) (5Ni) complex. A much improved synthesis of the starting material 7Ni by Vilsmeier-Haack formylation of [meso-tetraphenylchlorophinato]Ni(II) (8Ni) is also reported.     

二氢卟吩e6赖氨酸酰胺稳定性及电子光谱

采用密度泛函理论在B3LYP/6-31G^*水平上对二氢卟吩e6(简称e6)及设计的6个e6赖氨酸酰胺进行几何优化, 对优化构型用B3LYP/6-31G^**法进行单点计算, 并用含时密度泛函理论LSDA/6-31G^**方法计算电子吸收光谱. 结果表明, 赖氨酸的ε-NH₂与e6连接的酰胺更稳定, 其中, 15位的乙酰胺Y_ε最稳定. 形成赖氨酸酰胺改善了e6 的水溶性, 有利于药物吸收. 各e6 赖氨酸酰胺的前线轨道集中于二氢卟吩环, 由于连接酰胺基侧链的二氢卟吩环平面性有所下降, 前线轨道能隙略为升高, 最大电子吸收波长相对于e6 蓝移16-39 nm, 但仍处于光动力治疗窗口“600-900 nm”. 酰胺链的构象对吸收波长影响较大, Y_ε三个较稳定构象中, 酰胺链垂直于二氢卟吩环的Y_ε1和Y_ε2的二氢卟吩环平面性较好, 最大吸收波长比酰胺链与二氢卟吩环近似平面的Y_ε红移53、50nm, 三者平均值较e6红移18 nm.     

Synthesis of strapped zinc chlorophyll derivatives and their complexation with a single axial ligand

Zinc chlorins having a bridged moiety between 3- or 8- and 17-positions were designed as protected chlorophylls at one of π-faces, aiming at the investigation of asymmetric coordination ability towards the central metal. These strapped chlorins were synthesized by the cyclization of the dihydroxylated chlorins with dicarboxylic acid dichlorides under highly diluted conditions. The synthetic zinc chlorins complexed with pyridine as an axial ligand in benzene to form 5-coordinated species. The 1:1 binding constants determined by UV-vis titration method were smaller than those of the corresponding acyclic (unstrapped) compounds.     

Trifluoroacetyl-chlorin as a new chemosensor for alcohol/amine detection

Chlorophyll derivative possessing a trifluoroacetyl group at the 3-position was synthesized as a new chemosensor for alcohols and amines. Intense Q_y peak of the trifluoroacetyl-chlorin (701 nm in CHCl₃) showed blue shifts to 667 nm in MeOH and 665 nm in n-BuNH₂ due to the formation of the corresponding hemiacetal and hemiaminal with visible color changes. Thermodynamic parameters for the complexation between trifluoroacetyl-chlorin and n-BuNH₂ in CDCl₃ were determined to be ΔH = −48 kJ mol⁻¹ and ΔS = −147 J K⁻¹ mol⁻¹. Ratiometric fluorescence sensing of n-BuNH₂ in THF was also demonstrated.     

Synthesis of stereospecifically face-protected chlorophyll derivatives

A pair of zinc chlorins having a bridged moiety between the 3 and 13²-positions on the front or back side of its π-face were synthesized, and their asymmetric coordination ability towards pyridine was determined in benzene.