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.     

Optical method for monitoring of photodynamic inactivation of bacteria

Photodynamic inactivation is a new promising approach to treat bacterial infections. Usually, the evaluation of the efficacy of this method is done through time-consuming and labor-intensive microbiological test methods. This paper describes the development and implementation of an optical method to evaluate the photodynamic inactivation of bacteria based on non-invasive diffuse reflectance measurements. Five Staphylococcus aureus cultures and 15 mice have been used in this study. A skin lesion was created on the back of all animals, and it was contaminated with S. aureus (5.16 ± 0.013 log CFU/ml). Toluidine Blue O (c = 8.67 × 10 ^− 3 M) has been used as a photosensitiser agent. The bacterial cultures and animals were exposed to laser radiation (λ = 635 nm, P = 15 mW, DE = 8.654 J/cm²) for 20 min. The photodynamic inactivation of bacteria was monitored by acquiring the wounds’ reflection spectra at different time points and by microbiological exams on the bioptical material. The good correlation between the diffuse reflectance and colony-forming units demonstrates the value of this optical method based on diffuse reflectance measurements as a rapid technique to monitor photodynamic bacterial inactivation.     

Superoxide dismutase is upregulated in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> following protoporphyrin-mediated photodynamic inactivation and does not directly influence the response to photodynamic treatment

Background  

Staphylococcus aureus, a major human pathogen causes a wide range of disease syndromes. The most dangerous are methicillin-resistant S. aureus (MRSA) strains, resistant not only to all β-lactam antibiotics but also to other antimicrobials. An alarming increase in antibiotic resistance spreading among pathogenic bacteria inclines to search for alternative therapeutic options, for which resistance can not be developed easily. Among others, photodynamic inactivation (PDI) of S. aureus is a promising option. Photodynamic inactivation is based on a concept that a non toxic chemical, called a photosensitizer upon excitation with light of an appropriate wavelength is activated. As a consequence singlet oxygen and other reactive oxygen species (e.g. superoxide anion) are produced, which are responsible for the cytotoxic effect towards bacterial cells. As strain-dependence in photodynamic inactivation of S. aureus was observed, determination of the molecular marker(s) underlying the mechanism of the bacterial response to PDI treatment would be of great clinical importance. We examined the role of superoxide dismutases (Sod) in photodynamic inactivation of S. aureus as enzymes responsible for oxidative stress resistance.     

Attaching NorA efflux pump inhibitors to methylene blue enhances antimicrobial photodynamic inactivation of Escherichia coli and Acinetobacter baumannii in vitro and in vivo

Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en–MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en–MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity.     

Liquid crystal precursor system as a vehicle for curcumin-mediated photodynamic inactivation of oral biofilms

Curcumin has great potential as a photosensitizer, but it has low solubility in aqueous solutions. This study reports the antimicrobial efficacy of photodynamic inactivation (PDI) mediated by a curcumin-loaded liquid crystal precursor (LCP) on in situ dental biofilms. Thirty volunteers used intraoral devices containing enamel samples for 48 hours for biofilm formation. The samples were then removed from the device and treated either with LCP with 160 μM of curcumin plus illumination at 18 J/cm² (C + L+ group) or with LCP without curcumin in the dark (C – L − group). Following this, the biofilm from the samples was plated for quantifying the viable colonies at 37°C for 48 hours. Specific and nonspecific media were used for the presumptive isolation of Streptococcus mutans, Lactobacillus species/aciduric microorganisms, Candida species, and total microbiota. The C + L+ group showed a highly significant (P < .001) reduction in the log₁₀ (colony forming units/mL) values as compared to the C − L − group for all culture media. Hierarchical linear regression indicated that there may be predictors at individual volunteer level explaining the difference in the PDI efficacy among different individuals (P = .001). The LCP system retained curcumin and released it slowly and continuously, thus protecting the drug from photodegradation. LCP with curcumin is considered effective for the photoinactivation of dental biofilms, but the PDI efficacy may differ based on the host's individual characteristics.     

The Synergistic Antibacterial Properties of Glycinin Basic Peptide against Bacteria via Membrane Damage and Inactivation of Enzymes

This study investigated the antibacterial properties of glycinin basic peptide (GBP), a natural antibacterial component from soybean protein, against Staphylococcus aureus (S. aureus). The minimum inhibitory and bactericidal concentrations of GBP against S. aureus were 0.2 mg/mL and 0.8 mg/mL, respectively. Flow cytometry analysis manifested that GBP decreased the number of intact and normal cells. Higher concentrations of GBP induced more severe damage of the bacterial membrane; the maximal percentage of injured and dead cells was 93.8% with 0.8 mg/mL GBP. Electron microscopy imaging visually showed the morphological damage of S. aureus by GBP. Intracellular K⁺ leakage and the membrane depolarization of S. aureus further verified that GBP could destroy the bacterial membrane. Moreover, GBP decreased the activity of nonspecific esterase and ATPase of S. aureus in a concentration-dependent manner. These results demonstrated that GBP exhibited antibacterial properties against S. aureus via synergistic actions of damage to the cell membrane and inactivation of metabolic enzymes.

     

In vitro and in vivo responses of fungal biocontrol agents to gradient doses of UV-B and UV-A irradiation

Conidia of Beauveria bassiana and Metarhizium spp. smeared on glass slides were assayed for their responses to irradiation with weighted 312-nm UV-B and 365-nm UV-A at gradient doses of 0.005–1.1 and 1.0–18.0 J cm⁻², respectively. All inverted, sigmoid dose–survival trends showed good fit to a survival model (r ² ≥ 0.97), yielding respective UV-B LD₅₀s of 0.23–0.59 and 0.05–0.65 J cm⁻² for 24 B. bassiana and 36 Metarhizium isolates, and UV-A LD₅₀s of 2.78–10.46 J cm⁻² for 24 Metarhizium isolates. Myzus persicae apterae on detached leaves were sprayed with a concentrated spore suspension of B. bassiana or M. anisopliae, followed by exposure to the UV-B doses to cause 10–90% viability losses. These doses caused aphid mortality reductions as expected but affected neither spray-to-death period nor fungal growth on cadavers. The results highlight the merits of using UV-tolerant candidates and photoprotection measures in fungal formulations for pest control.     

Comparative Tolerances of Various <Emphasis Type="Italic">Beauveria bassiana</Emphasis> Isolates to UV-B Irradiation with a Description of a Modeling Method to Assess Lethal Dose

The tolerances of 20 Beauveria bassiana isolates derived from host insects worldwide to UV-B irradiation were assessed quantitatively in multi-dose bioassays. Conidial suspensions of the isolates smeared on glass slides were exposed to the gradient UV-B doses of 0.1–1.6 J cm⁻² (D), which generated from 0.75 to 10.17 min irradiation of weighted 312-nm wavelength at 2.0–2.61 mW cm⁻². Irradiated conidia were then incubated for 24 h at 25°C under saturated humidity. The ratio of germination at each dose over that in the blank control was defined as survival index (I _s). For all isolates, the I _s − D observations fit well with the survival model I _s = 1/[1 + exp(a + bD)] (0.94 ≤ r ² ≤ 0.99) generated widely spanned lethal doses of 0.154–0.928, 0.240–1.139, and 0.383–1.493 J cm⁻² for their losses of 50%, 75%, and 95% viabilities, respectively. These were far below the solar UV-B dose of 2.439 J cm⁻² measured in a sunny day during the summer. The large variation of UV-B tolerance among the isolates indicates a necessity to select UV-tolerant candidates for formulations applied to insect control during summer. The highly efficient bioassay method was developed to measure accurately the UV-B tolerances of fungal biocontrol agents as lethal doses.     

Lethal effects of high-intensity violet 405-nm light on Saccharomyces cerevisiae,Candida albicans,and on dormant and germinating spores of Aspergillus niger

This study assessed the effects of high-intensity violet light on selected yeast and mould fungi. Cell suspensions of Saccharomyces cerevisiae, Candida albicans, and dormant and germinating spores (conidia) of the mould Aspergillus niger were exposed to high-intensity narrow band violet light with peak output at 405 nm generated from a light-emitting diode (LED) array. All three fungal species were inactivated by the 405-nm light without a requirement for addition of exogenous photosensitiser chemicals. Of the fungal species tested, S. cerevisiae was most sensitive and dormant conidia of A. niger were most resistant to 405-nm light exposure. Five-log₁₀ colony forming units per millilitre (CFU ml^?1) reductions of the tested species required exposure doses of 288 J cm^?2 for S. cerevisiae, 576 J cm^?2 for C. albicans, and a much higher value of 2.3 kJ cm^?2 for dormant conidia of A. niger. During germination, A. niger conidia became more sensitive to 405-nm light exposure and sensitivity increased as germination progressed over an 8 h test period. Light exposure under aerobic and anaerobic conditions, together with results obtained using ascorbic acid as a scavenger of reactive oxygen species, revealed that 405-nm light inactivation in fungi involved an oxygen-dependent mechanism, as previously described in bacteria. The inactivation results achieved with yeast cells and fungal spores together with operational advantages associated with the use of a visible (nonultraviolet (UV)) light source highlight the potential of 405-nm light for fungal decontamination applications.     

Polyphenolic Antioxidants Efficiently Protect Urease from Inactivation by Ultrasonic Cavitation

Inactivation of urease (25 nM) in aqueous solutions (pH 5.0–6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm², 36–56°C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 W/cm², 36 or 56°C) has been characterized quantitatively, using first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(us), ultrasonic inactivation. Within the range from 1 nM to 10 M, propyl gallate (PG) decreases by approximately threefold the rate of LFUS-induced inactivation of urease (56°C), whereas resorcinol poly-2-disulfide stops this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36°C) or 10 nM (56°C). At 0.2–1.0 M, human serum albumin (HSA) increases the resistance of urease treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS–HSA), formed by conjugation of 1.0 nM GAPDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56°C).     

Trichophyton rubrum is Inhibited by Free and Nanoparticle Encapsulated Curcumin by Induction of Nitrosative Stress after Photodynamic Activation

Antimicrobial photodynamic inhibition (aPI) utilizes radical stress generated from the excitation of a photosensitizer (PS) with light to destroy pathogens. Its use against Trichophyton rubrum, a dermatophytic fungus with increasing incidence and resistance, has not been well characterized. Our aim was to evaluate the mechanism of action of aPI against T. rubrum using curcumin as the PS in both free and nanoparticle (curc-np) form. Nanocarriers stabilize curcumin and allow for enhanced solubility and PS delivery. Curcumin aPI, at optimal conditions of 10 μg/mL of PS with 10 J/cm² of blue light (417 ± 5 nm), completely inhibited fungal growth (p<0.0001) via induction of reactive oxygen (ROS) and nitrogen species (RNS), which was associated with fungal death by apoptosis. Interestingly, only scavengers of RNS impeded aPI efficacy, suggesting that curcumin acts potently via a nitrosative pathway. The curc-np induced greater NO^• expression and enhanced apoptosis of fungal cells, highlighting curc-np aPI as a potential treatment for T. rubrum skin infections.     

Use of Merocyanine 540 for Photodynamic Inactivation of Staphylococcus aureus Planktonic and Biofilm Cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 μg of MC 540 per ml and a light dose of 600 J/cm² or to 20 μg of MC 540 per ml and a light dose of 450 J/cm². A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

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.     

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.     

Combination of high‐frequency ultrasound and UV radiation of excilamp for surface disinfection

The combination of high‐frequency ultrasound (HFUS) and UV represents a new approach to disinfecting surfaces. This study aimed to examine the inactivation efficiency of HFUS (1.7 MHz) and monochromatic UV radiation of KrCl excilamp (222 nm) in a single and a sequential mode against Bacillus cereus cells and spores added to glass surfaces. When treated by UV only, cells at populations of 10³, 10⁴, and 10⁵ colony‐forming units (CFU)/cm² showed 100% disinfection at high doses up to 1760 mJ/cm². Spores at 10⁴ CFU/cm² were completely inactivated at a dose of 1170 mJ/cm². Treatment with aqueous aerosol (produced by HFUS) reduced cell counts by 100% within a 40‐min exposure, whereas it was ineffective in inactivating spores under these conditions. In a sequential mode, the contaminated surface was pretreated with the sonicated aqueous aerosol and subsequently irradiated with the excilamp. It was found that HFUS exposure times and UV doses for complete inactivation decreased by a factor of 2 and 6–7, respectively, compared to sole HFUS or UV. A portable apparatus for surface disinfection was designed. The combined HFUS/UV method may be a promising technique for rapid disinfection of microbially contaminated surfaces.     

The inhibitory effect of curcumin on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We investigated the effects of curcumin, the principal active compound of turmeric, on voltage-dependent K⁺ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Curcumin reduced the Kv current in a dose-dependent manner with an apparent K_d value of 1.07 ± 0.03 μM. Although curcumin did not alter the kinetics of Kv current activation, it predominantly accelerated the decay rate of channel inactivation. The association and dissociation rate constants of curcumin were 1.35 ± 0.05 μM^?1 s^?1 and 1.47 ± 0.17 s^?1, respectively. Curcumin did not alter the steady-state activation or inactivation curves. Application of train pulses (1 or 2 Hz) increased curcumin-induced blockade of the Kv current, and the recovery time constant also increased in the presence of curcumin suggesting, that the inhibitory action of Kv currents by curcumin was use-dependent. From these results, we concluded that curcumin inhibited vascular Kv current in a state-, time-, and use-dependent manner.     

Blue light (470 nm) effectively inhibits bacterial and fungal growth

Blue light (470 nm) LED antimicrobial properties were studied alone against bacteria and with or without the food grade photosensitizer, erythrosine (ERY) against filamentous fungi. Leuconostoc mesenteroides (LM), Bacillus atrophaeus (BA) or Pseudomonas aeruginosa (PA) aliquots were exposed on nutrient agar plates to Array 1 (AR1, 0·2 mW cm^?2) or Array 2 (AR2, 80 mW cm^?2), which emitted impure or pure blue light (0–300 J cm^?2), respectively. Inoculated control (room light only) plates were incubated (48 h) and colonies enumerated. The antifungal properties of blue light combined with ERY (11·4 and 22·8 μmol l^?1) on Penicillium digitatum (PD) and Fusarium graminearum (FG) conidia were determined. Conidial controls consisted of: no light, room light‐treated conidia and ERY plus room light. Light‐treated (ERY + blue light) conidial samples were exposed only to AR2 (0–100 J cm^?2), aliquots spread on potato dextrose agar plates, incubated (48 h, 30°C) and colonies counted. Blue light alone significantly reduced bacterial and FG viability. Combined with ERY, it significantly reduced PD viability. Blue light is lethal to bacteria and filamentous fungi although effectiveness is dependent on light purity, energy levels and microbial genus.

Significance and Impact of the Study

Light from two arrays of different blue LEDs significantly reduced bacterial (Leuconostoc mesenteroides, Bacillus atrophaeus and Pseudomonas aeruginosa) viabilities. Significant in vitro viability loss was observed for the filamentous fungi, Penicillium digitatum and Fusarium graminearum when exposed to pure blue light only plus a photosensitizer. F. graminearum viability was significantly reduced by blue light alone. Results suggest that (i) the amount of significant loss in bacterial viability observed for blue light that is pure or with traces of other wavelengths is genus dependent and (ii) depending on fungal genera, pure blue light is fungicidal with or without a photosensitizer.     

Reactivation of Giardia lamblia cysts after exposure to polychromatic UV light

Aims: In this study, we determined the ability of a promising alternative UV technology – a polychromatic emission from a medium‐pressure UV (MP UV) technology – to inhibit the reactivation of UV‐irradiated Giardia lamblia cysts. Methods and Results: A UV‐collimated beam apparatus was used to expose shallow suspensions of purified G. lamblia cysts in PBS (pH 7·2) or filtered drinking water to a low dose (1 mJ cm^?2) of MP UV irradiation. After UV irradiation, samples were exposed to two repair conditions (light or dark) and two temperature conditions (25°C or 37°C for 2–4 h). The inactivation of G. lamblia cysts by MP UV was very extensive, and c. 3 log₁₀ inactivation was achieved with a dose of 1 mJ cm^?2. Meanwhile, there was no apparent reactivation (neither in vivo nor in vitro) of UV‐irradiated G. lamblia under the conditions tested. Conclusion: The results of this study indicated that, unlike the traditional low‐pressure (LP) UV technology, an alternative UV technology (MP UV) could inhibit the reactivation of UV‐irradiated G. lamblia cysts even when the cysts were exposed to low UV doses. Significance and Impact of the Study: It appears that alternative UV technology has some advantages over the traditional LP UV technology in drinking water disinfection because of their high level of inactivation against G. lamblia cysts and also effective inhibition of reactivation in UV‐irradiated G. lamblia cysts.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.     

Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC₅₀ value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC₅₀ value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10⁻⁷ M⁻¹ s⁻¹ and 4.0 × 10⁻⁹ M⁻¹ s⁻¹ for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin–apigenin binding was 1:1 and binding the binding constant (K_d) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G₂/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α–β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.