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The heme protein cytochrome c (Cyt c) plays pivotal roles in cellular life and death processes. In the respiratory chain of mitochondria, it serves as an electron transfer protein, contributing to the proliferation of healthy cells. In the cell cytoplasm, it activates intrinsic apoptosis to terminate damaged cells. Insight into these mechanisms and...
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... are four main classes of Cyt c in mammalian organisms (I-IV), which are categorized based on the number of hemes, the position and identity of the axial iron ligands, and the reduction potentials [33,34]. The heme groups that can be present in Cyt c can be classified into types A-D depending on the substituents on the porphyrin ring ( Figure 7) [35]. Cyt c are made of the type C heme, hence their name. ...
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... Different research groups reported the delivery of bioactive cytochrome C via carriers such as liposomes and different types of nanoparticles [36][37][38][39][40][41]. Thus far, carrier-free delivery of cytochrome C remains rare, and nonspecific delivery is often encountered [42]. Recently, our group has developed several carrier-free strategies for selectively delivering bioactive proteins such as green fluorescent protein and ß-galactosidase, as well as small molecules, ...
... Different research groups reported the delivery of bioactive cytochrome C via carriers such as liposomes and different types of nanoparticles [36][37][38][39][40][41]. Thus far, carrier-free delivery of cytochrome C remains rare, and nonspecific delivery is often encountered [42]. Recently, our group has developed several carrier-free strategies for selectively delivering bioactive proteins such as green fluorescent protein and ß-galactosidase, as well as small molecules, into tumour cells by conjugating them with tumour-specific binding peptides against tumour biomarkers such as integrin and EGFR [43][44][45][46][47]. ...
Delivering bioactive proteins into cells without carriers presents significant challenges in biomedical applications due to limited cell membrane permeability and the need for targeted delivery. Here, we introduce a novel carrier-free method that addresses these challenges by chemically modifying proteins with an acid-responsive cell-penetrating peptide (CPP) for selective intracellular delivery within tumours. Cytochrome C, a protein known for inducing apoptosis, served as a model for intracellular delivery of therapeutic proteins for cancer treatment. The CPP was protected with 2,3-dimethyl maleic anhydride (DMA) and chemically conjugated onto the protein surface, creating an acid-responsive protein delivery system. In the acidic tumour microenvironment, DMA deprotects and exposes the positively charged CPP, enabling membrane penetration. Both in vitro and in vivo assays validated the pH-dependent shielding mechanism, demonstrating the modified cytochrome C could induce apoptosis in cancer cells in a pH-selective manner. These findings provide a promising new approach for carrier-free and tumour-targeted intracellular delivery of therapeutic proteins for a wide range of potential applications.
... (1,46) Cytochrome C, an electron carrier in the mitochondrial electron transport chain, acts as a cellular life-and-death decision maker molecule that regulates cellular energy supply and apoptosis. 47,48 Mitochondrial homeostasis is maintained by melatonin through its protective effect, which plays a critical role in the physiological processes of the cell, such that the defect in the mitochondria is considered the main cause of many pathological processes, including skin aging. (1,49) Melatonin and skin aging Circadian rhythm homeostasis is regulated by epidermal stem cells to maintain a healthy skin by managing the energy supply and cellular conditions for optimal protection and repair. ...
The ultraviolet radiation of the sun that reaches the earth is made up of ultraviolet A (95%) and ultraviolet B (5%). Exposure to ultraviolet radiation (UVR) is the main factor in photoaging. Chronic exposure to sunlight acts as an environmental stressor, leading to oxidative damage or stress. Oxidative damage stimulates the accumulation of free radicals, such as reactive oxygen species (ROS) or reactive nitrogen species (RNS) that are responsible for premature skin aging. Photoaged skin is characterized externally by irregular pigmentation, wrinkles, and reduced skin elasticity, and internally by the breakdown of dermal collagen and elastin. Free radicals can be scavenged and the skin can be protected from further oxidative damage by antioxidants. Melatonin is a hormone produced mainly by the pineal gland, as well as many other organs, including the skin. One of the functions of melatonin is exerted by the antioxidative melatoninergic system to prevent ultraviolet (UV)-induced skin photoaging. The aim of this review was to study the protective effect of melatonin on skin photoaging resulting from UVR exposure. The references were tracked using various databases, such as Google Scholar and PubMed with regard to publications in English for the last 5 years (2019-2023). Melatonin inhibits UVR-induced aging in multiple ways, such as protecting skin cells, binding free radicals, accelerating the activity of antioxidant enzymes, preventing increased mitochondrial membrane permeability, and assisting electron transport efficiency in the mitochondrial respiratory chain. Exogenous application of melatonin is usually by the oral route, but for localized effects on the skin, topical administration is recommended, with consideration of preparations with a better half-life and bioavailability. Understanding the protective antioxidant function of melatonin in UV-induced skin photoaging helps to optimize its application. The protective properties of melatonin against UVR-oxidative stress-induced photoaging will be further explored in this review.
... In breast tumors, there is a notable increase in the levels of cytochrome c, which is associated with tumor grade and aggressiveness [29]. Consequently, cytochrome c emerges as a potential diagnostic marker for cancer and a significant target for anticancer therapeutics [30]. Furthermore, the redox state of cytochromes within the mitochondria has been implicated in treatment resistance and unfavorable prognosis in cancer cases [30]. ...
... Consequently, cytochrome c emerges as a potential diagnostic marker for cancer and a significant target for anticancer therapeutics [30]. Furthermore, the redox state of cytochromes within the mitochondria has been implicated in treatment resistance and unfavorable prognosis in cancer cases [30]. ...
Triple negative breast cancer (TNBC) is a highly aggressive form of cancer. Detecting TNBC early is crucial for improving disease prognosis and optimizing treatment. Unfortunately, conventional imaging techniques fall short in providing a comprehensive differentiation of TNBC subtypes due to their limited sensitivity and inability to capture subcellular details. In this study, we present a multimodal imaging platform that integrates heavy water (D2O)-probed stimulated Raman scattering (DO-SRS), two-photon fluorescence (TPF), and second harmonic generation (SHG) imaging. This platform allows us to directly visualize and quantify the metabolic activities of TNBC subtypes at a subcellular level. By utilizing DO-SRS imaging, we were able to identify distinct levels of de novo lipogenesis, protein synthesis, cytochrome c metabolic heterogeneity, and lipid unsaturation rates in various TNBC subtype tissues. Simultaneously, TPF imaging provided spatial distribution mapping of NAD[P]H and flavin signals in TNBC tissues, revealing a high redox ratio and significant lipid turnover rate in TNBC BL2 (HCC1806) samples. Furthermore, SHG imaging enabled us to observe diverse orientations of collagen fibers in TNBC tissues, with higher anisotropy at the tissue boundary compared to the center. Our multimodal imaging platform offers a highly sensitive and subcellular approach to characterizing not only TNBC, but also other tissue subtypes and cancers.
... In this regards, the proapoptotic properties of cyt c have been recently exploited with the aim to use the protein as an anticancer agent [108]. Different carrier scaffolds have Finally, the activation of proapoptotic events, including the release of cyt c after the CL peroxidation, may be of relevance for the development of efficient and specific therapies against cancer [107]. ...
... In this regards, the proapoptotic properties of cyt c have been recently exploited with the aim to use the protein as an anticancer agent [108]. Different carrier scaffolds have been designed for cyt c delivery into cancer cells (especially nanoparticles and conjugation to other proteins). ...
In the cell cytochrome, c performs different functions depending on the environment in which it acts; therefore, it has been classified as a multifunction protein. When anchored to the outer side of the inner mitochondrial membrane, native cytochrome c acts as a Schweitzer-StennerSchweitzer-Stenner that transfers electrons from cytochrome c reductase to cytochrome c oxidase in the respiratory chain. On the other hand, to interact with cardiolipin (one of the phospholipids making up the mitochondrial membrane) and form the cytochrome c/cardiolipin complex in the apoptotic process, the protein reorganizes its structure into a non-native state characterized by different asymmetry. The formation of the cytochrome c/cardiolipin complex is a fundamental step of the apoptotic pathway, since the structural rearrangement induces peroxidase activity in cytochrome c, the subsequent permeabilization of the membrane, and the release of the free protein into the cytoplasm, where cytochrome c activates the apoptotic process. Apoptosis is closely related to the pathogenesis of neoplastic, neurodegenerative and cardiovascular diseases; in this contest, the biosynthesis and remodeling of cardiolipin are crucial for the regulation of the apoptotic process. Since the role of cytochrome c as a promoter of apoptosis strictly depends on the non-native conformation(s) that the protein acquires when bound to the cardiolipin and such event leads to cytochrome c traslocation into the cytosol, the structural and functional properties of the cytochrome c/cardiolipin complex in cell fate will be the focus of the present review.
Intracellular protein delivery shows promise as a selective and specific approach to cancer therapy. However, a major challenge is posed by delivering proteins into the target cells. Despite the development of nanoparticle (NP)-based approaches, a versatile and biocompatible delivery system that can deliver active therapeutic cargo into the cytosol while escaping endosome degradation remains elusive. In order to overcome these challenges, a polymeric nanocarrier was prepared using cationic dextrin (CD), a biocompatible and biodegradable polymer, to encapsulate and deliver cytochrome C (Cyt C), a therapeutic protein. The challenge of endosomal escape of the nanoparticles was addressed by co-delivering the synthesized NP construct with chloroquine, which enhances the endosomal escape of the therapeutic protein. No toxicity was observed for both CD NPs and chloroquine at the concentration tested in this study. Spectroscopic investigations confirmed that the delivered protein, Cyt C, was structurally and functionally active. Additionally, the delivered Cyt C was able to induce apoptosis by causing depolarization of the mitochondrial membrane in HeLa cells, as evidenced by flow cytometry and microscopic observations. Our findings demonstrate that an engineered delivery system using CD NPs is a promising platform in nanomedicine for protein delivery applications.
Multifunctional gold nanoparticles (AuNPs) are of great interest, owing to their vast potential for use in many areas including sensing, imaging, delivery, and medicine. A key factor in determining the biological activity of multifunctional AuNPs is the quantification of surface conjugated molecules. There has been a lack of accurate methods to determine this for multifunctionalized AuNPs. We address this limitation by using a new method based on the deconvolution and Levenberg-Marquardt algorithm fitting of UV-visible absorption spectrum to calculate the precise concentration and number of cytochrome C (Cyt C) and zinc porphyrin (Zn Porph) bound to each multifunctional AuNP. Dynamic light scattering (DLS) and zeta potential measurements were used to confirm the functionalization of AuNPs with Cyt C and Zn Porph. Transmission electron microscopy (TEM) was used in conjunction with UV-visible absorption spectroscopy and DLS to identify the AuNP size and confirm that no aggregation had taken place after functionalization. Despite the overlapping absorption bands of Cyt C and Zn Porph, this method was able to reveal a precise concentration and number of Cyt C and Zn Porph molecules attached per AuNP. Furthermore, using this method, we were able to identify unconjugated molecules, suggesting the need for further purification of the sample. This guide provides a simple and effective method to quickly quantify molecules bound to AuNPs, giving users valuable information, especially for applications in drug delivery and biosensors.
As one of the frequent malignancies, breast cancer (BCa) is the foremost reason for cancer-related deaths among women. The role of Human papillomavirus (HPV) in chemoresistance has rarely been investigated in previous studies. The current study sets out to the possible role of HPV in BCa chemoresistance. In this research, 90 BCa tissue and 33 normal breast tissue were collected. We evaluated the presence of the HPV genome along with the viral (E2, E6, E7) and cellular gene expression associated with cell resistance to death. Statically significant differences in the prevalence of HPV between the BCa group (25.2% or 23/90) and the control group (21.8% or 7/32) were not found. HPV-16 and HPV-18 genotypes were the abundant HPV genotypes. Resistance to the Adriamycin-Cyclophosphamide (AC), paclitaxel regimen was elevated in the HPV- group (56/70) in comparison to the HPV+ group (14/70). Nevertheless, there was no significant difference in the prevalence of resistance to AC + paclitaxel + triple-negative breast cancer combination therapy between the HPV+ group (9/20) and in the HPV- group (11/20). In the BCa group in contrast to the control group, the expression level of Bcl-2, BCL-XL, and c-IAP2 demonstrated a significant decrease, while, the expression level of cytochrome C and caspase 3 was significantly increased. This study suggests that HPV infection might contribute to BCa chemoresistance through disrupt cellular genes involved in cell death.
Cytochrome c (cyt c) is an electron transporter of the mitochondrial respiratory chain. Upon permeabilization of the mitochondrial outer membrane, cyt c is released into the cytoplasm, where it triggers the intrinsic pathway of apoptosis. Cytoplasmic cyt c can further reach the bloodstream. Apoptosis inhibition is one of the hallmarks of cancer and its induction in tumors is a widely used therapeutic approach. Apoptosis inhibition and induction correlate with decreased and increased serum levels of cyt c, respectively. The quantification of cyt c in the serum is useful in the monitoring of patient response to chemotherapy, with potential prognosis value. Several highly sensitive biosensors have been developed for the quantification of cyt c levels in human serum. Moreover, the delivery of exogenous cyt c to the cytoplasm of cancer cells is an effective approach for inducing their apoptosis. Similarly, several protein-based and nanoparticle-based systems have been developed for the therapeutic delivery of cyt c to cancer cells. As such, cyt c is a human protein with promising value in cancer prognosis and therapy. In addition, its thermal stability can be extended through PEGylation and ionic liquid storage. These processes could contribute to enhancing its therapeutic exploitation in clinical facilities with limited refrigeration conditions. Here, I discuss these research lines and how their timely conjunction can advance cancer therapy and prognosis.
Protein therapeutics are of widespread interest due to their successful performance in the current pharmaceutical and medical fields, even though their broad applications have been hindered by the lack of an efficient intracellular delivery approach. Herein, we fabricated an active-targeted dual pH-responsive delivery system with favorable tumor cell entry augmented by extracellular pH-triggered charge reversal and tumor receptor targeting and pH-controlled endosomal release in a traceless fashion. As a traceable model protein, the enhanced green fluorescent protein (eGFP) bearing a nuclear localization signal was covalently coupled with a pH-labile traceless azidomethyl-methylmaleic anhydride (AzMMMan) linker followed by functionalization with different molar equivalents of two dibenzocyclooctyne-octa-arginine-cysteine (DBCO-R8C)-modified moieties: polyethylene glycol (PEG)-GE11 peptide for epidermal growth factor receptor-mediated targeting and melittin for endosomal escape. The cationic melittin domain was masked with tetrahydrophthalic anhydride revertible at mild acidic pH 6.5. At the optimally balanced ratio of functional units, the on-demand charge conversion at tumoral extracellular pH 6.5 in combination with GE11-mediated targeting triggered enhanced electrostatic cellular attraction by the R8C cell-penetrating peptides and melittin, as demonstrated by strongly enhanced cellular uptake. Successful endosomal release followed by nuclear localization of the eGFP cargo was obtained by taking advantage of melittin-mediated endosomal escape and rapid traceless release from the AzMMMan linker. The effectiveness of this multifunctional bioresponsive system suggests a promising strategy for delivery of protein drugs toward intracellular targets. A possible therapeutic relevance was indicated by an example of cytosolic delivery of cytochrome c initiating the apoptosis pathway to kill cancer cells.
The interaction between cytochrome c (Cyt) and potential vanadium drugs, formed by 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (dhp) and maltolate (ma), was studied by ElectroSpray Ionization-Mass Spectrometry (ESI-MS). Since under physiological conditions redox processes are possible, the binding of the complexes in the oxidation state +IV and + V, [VIVO(dhp)2], [VIVO(ma)2], [VVO2(dhp)2]⁻ and [VVO2(ma)2]⁻, was examined. In all systems VIV,V–L–Cyt adducts are observed, their formation depending on V oxidation state, ligand and metal concentration. The larger stability of vanadium(IV) than vanadium(V) complexes favors the interaction of the moieties VIVOL2 and VIVOL⁺ with VIV, while with VV adducts with VVO2L and VVO2⁺ ion are observed. The analysis of the protein structure suggests that Glu4, Glu21, Asp50, Glu62, Glu66 and Glu104 are the most plausible candidates for monodentate coordination, while the couples (Asp2, Glu4), (Glu92, Glu93) and (His33, Glu104) for bidentate binding. The values of E1/2 for [VIVO(dhp)2] and [VIVO(ma)2], measured by cyclic voltammetry (CV), 0.53 V and 0.60 V vs. standard hydrogen electrode, indicate that an oxidation of VIV to VV is possible. The presence of a protein can alter the redox reactions and stabilize one of the states, VIV or VV. Overall, the data reinforce the conclusion that, for V drugs, the biotransformation is fundamental to explain their biological action and the analysis should not be limited to the ligand exchange and hydrolysis but also include the redox processes, and that a mixture of VIV and VV species, VIV,V–L–Protein and VIV,V–Protein, could be responsible of the pharmacological effects.
