Synthesis and Properties of O- and S-Glycosylated Derivatives of Pyropheophorbide a

New O- and S-glycosylated derivatives of pyropheophorbide awere synthesized in the context of the design of photosensitizers for photodynamic cancer therapy. The resulting amphiphilic conjugates were found to be sufficiently water-soluble and suitable for the study of the photosensitizer penetration and accumulation in tumors.     

The effects of adriamycin on E-cadherin mediated cell-cell adhesion and apoptosis during early kidney development

Abstract

Adriamycin (ADR) is strongly teratogenic. We investigated the effects of ADR on apoptosis and the intensity of E-cadherin expression in developing kidneys. An experimental group of rats was given 2 mg/kg/day ADR on days 6?9 of gestation and a control group was given saline on the same schedule. Embryos were decapitated on days 13, 15, 17 and 19 of gestation, and processed and embedded in paraffin for routine light microscopy. Kidney specimens were stained with hematoxylin and eosin or periodic acid-Schiff, or immunostained for E-cadherin. Apoptosis was assessed using the TUNEL method. Weight loss and developmental deficiency were determined in embryos of the experimental group. ADR damaged or destroyed tubule epithelial cells, which caused apparent dilatation of the tubule lumen. Also, the brush borders of proximal tubules were damaged and glomerular spaces were dilated. ADR caused apoptosis of kidney tissue by days 15, 17 and 19 of development and E-cadherin expression was up-regulated during kidney development compared to controls. We found that ADR can cause apoptosis and increased E-cadherin expression in the developing rat kidney. E-cadherin expression and apoptosis may contribute to the development of ADR nephrotoxicity.     

Biochemical studies on the potentiation of antitumor activity of cisplatin by adriamycin

The RNA synthesis in vitro by E. coli RNA polymerase was found to be highly sensitive to cis-diamminedichloroplatinum (cisplatin) inhibition. The degree of inhibition was in proportion to the length of time of template preincubation with cisplatin, suggesting that cisplatin-template binding was involved in the inhibition of RNA polymerase. The effect of adriamycin on this inhibition was studied and it was found that adriamycin significantly enhanced the inhibitory effect of cisplatin and that the total effect was greater than the sum of the effects of each drug used individually. This synergistic effect was not observed when the effect of the combination of adriamycin and cisplatin on in vitro DNA synthesis was studied.     

Amphiphilic tetracationic porphyrins are exceptionally active antimicrobial photosensitizers: In vitro and in vivo studies with the free‐base and Pd‐chelate

Antimicrobial photodynamic inactivation (aPDI) employs the combination of nontoxic photosensitizing dyes and visible light to kill pathogenic microorganisms regardless of drug‐resistance, and can be used to treat localized infections. A meso‐substituted tetra‐methylpyridinium porphyrin with one methyl group replaced by a C12 alkyl chain (FS111) and its Pd‐derivative (FS111‐Pd) were synthesized and tested as broad‐spectrum antimicrobial photosensitizers when excited by blue light (5 or 10 J/cm²). Both compounds showed unprecedented activity, with the superior FS111‐Pd giving 3 logs of killing at 1 nM, and eradication at 10 nM for Gram‐positive methicillin‐resistant Staphylococcus aureus. For the Gram‐negative Escherichia coli, both compounds produced eradication at 100 nM, while against the fungal yeast Candida albicans, both compounds produced eradication at 500 nM. Both compounds could be categorized as generators of singlet oxygen (Φ_Δ = 0.62 for FS111 and 0.71 for FS111‐Pd). An in vivo study was carried out using a mouse model of localized infection in a partial thickness skin abrasion caused by bioluminescent Gram‐negative uropathogenic E. coli. Both compounds were effective in reducing bioluminescent signal in a dose‐dependent manner when excited by blue light (405 nm), but aPDI with FS111‐Pd was somewhat superior both during light and in preventing recurrence during the 6 days following PDT.     

Comparative in vivo sensitizing efficacy of porphyrin and chlorin dimers joined with ester,ether, carbon–carbon or amide bonds

The anti-cancer activity of dimers joined with ether, ester or carbon–carbon bonds by photodynamic therapy (PDT) was compared by using DBA/2 mice transplanted with SMT/F tumors. Dimers with ether and carbon–carbon linkages were found to be more effective than those linked with ester bonds. Variation of the substituents at peripheral positions made a significant difference in in vivo efficacy. Among the ether and carbon–carbon linked dimers, the divinyl analogs were found to be most effective. The preliminary in vivo results also suggest that the position(s) of the hydrophilic substituents in the molecules make a remarkable difference in photosensitizing activity. An unsymmetrical dimer with an amide linkage, obtained from 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) was found to be less effective than HPPH.     

Two combined photosensitizers: a goal for more effective photodynamic therapy of cancer

Photodynamic therapy (PDT) is a clinically approved therapeutic modality for the treatment of diseases characterized by uncontrolled cell proliferation, mainly cancer. It involves the selective uptake of a photosensitizer (PS) by neoplastic tissue, which is able to produce reactive oxygen species upon irradiation with light, leading to tumor regression. Here a synergistic cell photoinactivation is reported based on the simultaneous administration of two PSs, zinc(II)-phthalocyanine (ZnPc) and the cationic porphyrin meso-tetrakis(4-N-methylpyridyl)porphine (TMPyP) in three cell lines (HeLa, HaCaT and MCF-7), using very low doses of PDT. We detected changes from predominant apoptosis (without cell detachment) to predominant necrosis, depending on the light dose used (2.4 and 3.6 J/cm², respectively). Analysis of changes in cytoskeleton components (microtubules and F-actin), FAK protein, as well as time-lapse video microscopy evidenced that HeLa cells were induced to undergo apoptosis, without losing adhesion to the substrate. Moreover, 24 h after intravenous injection into tumor-bearing mice, ZnPc and TMPyP were preferentially accumulated in the tumor area. PDT with combined treatment produced significant retardation of tumor growth. We believe that this combined and highly efficient strategy (two PSs) may provide synergistic curative rates regarding conventional photodynamic treatments (with one PS alone).     

Inhibition of plasma membrane NADH dehydrogenase by adriamycin and related anthracycline antibiotics

Doxorubicin (adriamycin) is cytotoxic to cells, but the biochemical basis for this effect is unknown, although intercalation with DNA has been proposed. This study suggests that the cytotoxicity of this drug may be due to inhibition of the plasma membrane redox system, which is involved in the control of cellular growth. Concentrations between 10^–6–10^–7 M adriamycin inhibit plasma membrane redox reactions >50%. AD32, a form of adriamycin which does not intercalate with DNA, but is cytotoxic, also inhibits the plasma membrane redox system. Thus, the cytotoxic effects of adriamycin, which limit its use as a drug, may be based on the inhibition of a transplasma membrane dehydrogenase involved in a plasma membrane redox system.     

Synthesis and spectral luminescent characteristics of the porphyrin complexes with the platinum group metals

The syntheses of complexes of natural and synthetic porphyrins with Pt, Pd, Rh, and Ru are reported. Their electronic absorption spectra, phosphorescence spectra, and lifetimes at room temperature both in the presence and in the absence of oxygen were studied. Variations in the nature of the central metal atom and in the substituents in pyrrol and phenyl rings allow the obtaining of metalloporphyrins phosphorescing at room temperature with various phosphorescence excitation and emission spectra.     

Propionate precursors in the biosynthesis of daunomycin and adriamycin: A 13C nuclear magnetic resonance study

A ¹³C-NMR study of the biosynthesis of daunomycin adriamycin from propionate[1-¹³C] has been carried out in cultures of Streptomyces peucetius var. caesius. Results give direct support for the postulate that a propionate ‘starter’ is involved in the biosynthesis of both metabolites.     

Assessment of new cationic porphyrin binding to plasma proteins by planar microarray and spectroscopic methods

Porphyrins have a unique aromatic structure determining particular photochemical properties that make them promising photosensitizers for anticancer therapy. Previously, we synthesized a set of artificial porphyrins by modifying side-chain functional groups and introducing different metals into the core structure. Here, we have performed a comparative study of the binding properties of 29 cationic porphyrins with plasma proteins by using microarray and spectroscopic approaches. The porphyrins were noncovalently immobilized onto hydrogel-covered glass slides and probed to bio-conjugated human and bovine serum albumins, as well as to human hemoglobin. The signal detection was carried out at the near-infrared fluorescence wavelength (800?nm) that enabled the effect of intrinsic visible wavelength fluorescence emitted by the porphyrins tested to be discarded. Competition assays on porphyrin microarrays indicated that long-chain fatty acids (FAs) (palmitic and stearic acids) decrease porphyrin binding to both serum albumin and hemoglobin. The binding affinity of different types of cationic porphyrins for plasma proteins was quantitatively assessed in the absence and presence of FAs by fluorescent and absorption spectroscopy. Molecular docking analysis confirmed results that new porphyrins and long-chain FAs compete for the common binding site FA1 in human serum albumin and meso-substituted functional groups in porphyrins play major role in the modulation of conformational rearrangements of the protein.     

Antimicrobial activity of amphiphilic nanomicelles loaded with curcumin against Pseudomonas aeruginosa alone and activated by blue laser light

The aim of this work was to assess the antimicrobial efficacy on Pseudomonas aeruginosa of nanomicelles loaded with curcumin (CUR) alone and activated by blue laser light in an antimicrobial photodynamic therapy (APDT) approach. First, free CUR in liquid suspension and loaded in three amphiphilic nanomicelles (CUR-DAPMA, CUR-SPD and CUR-SPM) were tested both on bacteria and keratinocytes. While free CUR exerted limited efficacy showing moderate cytotoxicity, a strong inhibition of bacterial growth was obtained using all three nanosystems without toxicity on eukaryotic cells. CUR-SPM emerged as the most effective, and was therefore employed in APDT experiments. Among the three sublethal blue laser (λ 445 nm) protocols tested, the ones characterized by a fluence of 18 and 30 J/cm² further decreased the antimicrobial concentration to 50 nM. The combination of blue laser APDT with CUR-SPM nanomicelles results in an effective synergistic activity that represents a promising novel therapeutic approach on resistant species.     

Improvement of tumor targeting and antitumor activity by a disulphide bond stabilized diabody expressed in Escherichia coli

We have generated an anti-Pgp/anti-CD3 diabody which can effectively inhibit the growth of multidrug-resistant human tumors. However, the two chains of the diabody are associated non-covalently and are therefore capable of dissociation. Cysteine residues were introduced into the V-domains to promote disulphide cross-linking of the dimer as secreted by Escherichia coli. Compared with the parent diabody, the ds-Diabody obtained was more stable in human serum at 37°C, without loss of affinity or cytotoxicity activity in vitro. Furthermore, the ds-Diabody showed improved tumor localization and a twofold improved antitumor activity over the parent diabody in nude mice bearing Pgp-overexpressing K562/A02 xenografts. Our data demonstrate that ds-Diabody may be more useful in therapeutic applications than the parent diabody.     

First success of catalytic epoxidation of olefins by an electron-rich iron(III) porphyrin complex and H2O2: imidazole effect on the activation of H2O2 by iron porphyrin complexes in aprotic solvent

An electron-rich iron(III) porphyrin complex (meso-tetramesitylporphinato)iron(III) chloride [Fe(TMP)Cl], was found to catalyze the epoxidation of olefins by aqueous 30% H₂O₂ when the reaction was carried out in the presence of 5-chloro-1-methylimidazole (5-Cl-1-MeIm) in aprotic solvent. Epoxides were the predominant products with trace amounts of allylic oxidation products, indicating that Fenton-type oxidation reactions were not involved in the olefin epoxidation reactions. cis-Stilbene was stereospecifically oxidized to cis-stilbene oxide without giving isomerized trans-stilbene oxide product, demonstrating that neither hydroperoxy radical (HOO·) nor oxoiron(IV) porphyrin [(TMP)Fe^IV=O] was responsible for the olefin epoxidations. We also found that the reactivities of other iron(III) porphyrin complexes such as (meso-tetrakis(2,6-dichlorophenyl)porphinato)iron(III) chloride [Fe(TDCPP)Cl], (meso-tetrakis(2,6-difluorophenyl)porphinato)iron(III) chloride [Fe(TDFPP)Cl], and (meso-tetrakis(pentafluorophenyl)porphinato)iron(III) chloride [Fe(TPFPP)Cl] were significantly affected by the presence of the imidazole in the epoxidation of olefins by H₂O₂. These iron porphyrin complexes did not yield cyclohexene oxide in the epoxidation of cyclohexene by H₂O₂ in the absence of 5-Cl-1-MeIm in aprotic solvent; however, addition of 5-Cl-1-MeIm to the reaction solutions gave high yields of cyclohexene oxide with the formation of trace amounts of allylic oxidation products. We proposed, on the basis of the results of mechanistic studies, that the role of the imidazole is to decelerate the O–O bond cleavage of an iron(III) hydroperoxide porphyrin (or H₂O₂–iron(III) porphyrin adduct) and that the intermediate transfers its oxygen to olefins prior to the O–O bond cleavage.     

In vitro evaluation of photodynamic activity of methylene blue against Trichophyton verrucosum azole-susceptible and -resistant strains

The intense search for the “Holy Grail” of antifungal therapy can be observed today. The searches are not limited only to discovery of potential antifungal drugs, but also new therapeutic strategies involving the use of chemosensitizers to achieve synergistic effect or physicochemical factors inducing stress conditions in fungal cells. In this study was examined in vitro effectiveness of photodynamic antifungal strategy with methylene blue using a light beam with a wavelength equal to 635 nm toward the Trichophyton verrucosum susceptible and itraconazole- and/or fluconazole-resistant strains. Methylene blue used at concentration equal to 5 μg/mL and in the presence of 40 J/cm² of light energy showed fungicidal effect toward the susceptible strains. However, for azole-resistant isolates, only the energy dose equal to 60 J/cm² at 5 μg/mL of methylene blue allowed to kill the pathogen. This study confirms that methylene blue induced by red light has a definite inhibitory effect on zoophilic dermatophytes.     

Synergistic antitumor effect of anti-PD-L1 combined with oxaliplatin on a mouse tumor model

Oxaliplatin (OXP) can change tumor microenvironment from immune-suppressive toward the immune-favorable condition. Almost all of the antitumor agents cannot totally cure cancer as monotherapy. So the current focus of cancer research became combining therapy using different treatment regimen, especially chemotherapy with checkpoint blockers. In this study, we assessed the activity of combining regimen using anti-PD-L1 with OXP in CT26 tumor-bearing BALB/c mice. We further analyzed the immune cell phenotypes in tumor site, lymph nodes, and spleen by flow cytometry analysis. Our study showed that combination therapy with OXP and anti-PD-L1 significantly increased survival in vivo and inhibited tumor growth of tumor-bearing mice. Inconsistent with better antitumor activity, our combination therapy led to an increase in tumor-infiltrating activated CD8+ T cells. In draining lymph nodes and spleen, regulatory T cells decreased significantly. Mice receiving either anti-PD-L1 or OXP alone had a larger tumor and lower survival rate in comparison with combination therapy receiving group. The time and order of administration of each component of the combination therapy affected antitumor response.     

黄芪注射液对阿霉素性心肌细胞凋亡、内质网应激与缝隙连接蛋白表达的影响

目的：研究黄芪注射液对阿霉素（ADR）所致心肌病大鼠心肌细胞凋亡、内质网应激与缝隙连接蛋白表达的影响。方法：36只Wistar雄性大鼠随机分为3组（n=12）：对照组、ADR组及黄芪注射液组。对照组腹腔注射0.9% Nacl （10 ml/kg体重）;ADR组腹腔注射ADR 2 mg/kg体重;黄芪注射液组在每次腹腔注射ADR 2 mg/kg体重的同时,注射黄芪注射液10 g/kg体重,每周注射1次,共注射3次。实验第7周末,3组大鼠行心脏彩超检测左室舒张末期内径、左室收缩末期内径及左室射血分数;处死大鼠后取左心室组织行HE、Masson、醋酸铀及柠檬酸铅染色,于光镜及透射电镜下观察心肌病理及超微结构改变;采用TUNEL法检测大鼠心肌细胞凋亡,用免疫组化技术检测大鼠心肌细胞缝隙连接蛋白Cx43及p-Cx43表达,采用real time PCR检测大鼠心肌细胞内质网应激伴侣蛋白Grp78,ATF-4及CHOP表达。结果：与对照组比较,ADR组大鼠LVEDD、LVESD增大,LVEF减少;心肌纤维排列紊乱,心肌纤维间质水肿,大量淋巴细胞浸润;线粒体肿胀、破坏,呈空泡样;心肌细胞凋亡数明显增多（P<0.01）;内质网应激相关蛋白Grp78、ATF-4及CHOP表达明显增高（P<0.01）;缝隙连接蛋白Cx43表达减少,而p-Cx43表达增多。与ADR组比较,黄芪注射液组大鼠LVEDD、LVESD减少,LVEF增加;心肌病理及超微结构明显改善,同时心肌细胞凋亡数明显减少（P<0.01）;内质网应激伴侣蛋白Grp78、ATF-4及CHOP表达明显减少（P<0.01）;缝隙连接蛋白Cx43表达增多,而p-Cx43减少。结论：黄芪注射液可有效改善阿霉素导致的心肌损伤,其机制可能与黄芪注射液抑制ADR诱导的内质网应激及缝隙连接蛋白磷酸化有关。