Attenuating mitochondrial dysfunction and morphological disruption with PT320 delays dopamine degeneration in MitoPark mice.

BACKGROUND: Mitochondria are essential organelles involved in cellular energy production. Changes in mitochondrial function can lead to dysfunction and cell death in aging and age-related disorders. Recent research suggests that mitochondrial dysfunction is closely linked to neurodegenerative diseases. Glucagon-like peptide-1 receptor (GLP-1R) agonist has gained interest as a potential treatment for Parkinson's disease (PD). However, the exact mechanisms responsible for the therapeutic effects of GLP-1R-related agonists are not yet fully understood. METHODS: In this study, we explores the effects of early treatment with PT320, a sustained release formulation of the GLP-1R agonist Exenatide, on mitochondrial functions and morphology in a progressive PD mouse model, the MitoPark (MP) mouse. RESULTS: Our findings demonstrate that administration of a clinically translatable dose of PT320 ameliorates the reduction in tyrosine hydroxylase expression, lowers reactive oxygen species (ROS) levels, and inhibits mitochondrial cytochrome c release during nigrostriatal dopaminergic denervation in MP mice. PT320 treatment significantly preserved mitochondrial function and morphology but did not influence the reduction in mitochondria numbers during PD progression in MP mice. Genetic analysis indicated that the cytoprotective effect of PT320 is attributed to a reduction in the expression of mitochondrial fission protein 1 (Fis1) and an increase in the expression of optic atrophy type 1 (Opa1), which is known to play a role in maintaining mitochondrial homeostasis and decreasing cytochrome c release through remodeling of the cristae. CONCLUSION: Our findings suggest that the early administration of PT320 shows potential as a neuroprotective treatment for PD, as it can preserve mitochondrial function. Through enhancing mitochondrial health by regulating Opa1 and Fis1, PT320 presents a new neuroprotective therapy in PD.

Herbal Melanin Inhibits Real-Time Cell Proliferation, Downregulates Anti-Apoptotic Proteins and Upregulates Pro-Apoptotic p53 Expression in MDA-MB-231 and HCT-116 Cancer Cell Lines.

Background and Objectives: Cancer is the second-most-important deadly disease in the world, leading to severe socioeconomic consequences and posing a public threat. Consequently, breast and colorectal cancers are significant cancer types that affect women and men more commonly, respectively. Treatment failure or recurrent diseases frequently occur due to resistance, in addition to the side effects of the currently available anticancer agents. Therefore, in this study, herbal melanin anticancer activity was investigated against human breast adenocarcinoma (MDA-MB-231) and human colorectal (HCT 116) cell proliferation and the expression of downregulated anti-apoptotic proteins and upregulated pro-apoptotic p53. Materials and Methods: MDA-MB-231 and HCT 116 cells were monitored for their real-time proliferation properties using Xcelligence. Herbal melanin of various concentrations significantly inhibited MDA-MB-231 and HCT 116 cell proliferation. Then, the expression of proapoptotic and anti-apoptotic proteins such as p53, Bcl-2 and Bcl-xl was studied using Western blotting. Results: The Bcl-2 and Bcl-xl expressions were downregulated, while the p53 expression was upregulated after treatment with herbal melanin. Similarly, the expression of apoptotic proteins such as Bcl-2, Bcl-xl, XIAP, Survivin, Bid, Bax, p53, Cytochrome C, PARP genes and mRNA was studied after herbal melanin treatment using real-time PCR, which revealed the downregulation of Bcl-2, Bcl-xl, XIAP and Survivin and the upregulation of Bid, Bax, p53, Cytochrome C and PARP apoptotic protein expression. Also, caspase 3 and 9 expressions were monitored after the treatment with herbal melanin, which revealed the upregulation of both the MDA-MB-231 and HCT 116 cell types. Conclusions: Overall, herbal melanin can be used as an alternative anticancer agent against the MDA-MB-231 and HCT 116 cell types.

Pinus massoniana needle extracts attenuate oxidative stress injury in cerebral ischemia reperfusion rats by regulating JNK3/caspase-3 signal transduction.

OBJECTIVE: To investigate the effect and mechanism of Pinus massoniana needle extracts (PNE) on oxidative stress injury in cerebral ischemia reperfusion rats. METHODS: The SD male rats were randomly divided into sham group, model control group, Edaravone (3 mg/kg) group, PNE low-dose (200 mg/kg), medium-dose (400 mg/kg) and high-dose (800 mg/kg) groups. PNE was administered by gavage for 7 d before modeling and 6 h after modeling in PNE treatment groups; Edaravone was given by intraperitoneal injection 7 d before modeling and 6 h after reperfusion. The rat model of cerebral ischemia reperfusion injury was established by middle cerebral artery occlusion method. After 24 h of reperfusion, the neurological deficit score, brain water content and cerebral infarction volume of rats were measured. The pathological changes of cerebral cortex and hippocampus were observed by HE staining, and the number of normal nerve cells was counted. The apoptosis rate of neurons in cerebral cortex was detected by TUNEL method. The content of nitric oxide (NO), malondialdehyde (MDA) and superoxide dismutase (SOD) activity in ischemic brain tissue were detected. The protein expression of c-Jun N-terminal kinase (JNK) 3, phosphorylated JNK3 (p-JNK3), B-cell lymphoma protein(Bcl) -2, Bcl-2 associated X (Bax), cytochrome C and caspase-3 in cerebral cortex were detected by Western blotting method. RESULTS: Compared with the model control group, the behavioral score, brain water content and cerebral infarction volume in PNE groups were significantly reduced (all P<0.05), the pathological damage of cerebral cortex and hippocampal CA1 area was significantly alleviated, and the number of normal nerve cells in ischemic cortex and hippocampal CA1 area was increased (all P<0.05). The medium-dose PNE group had the best effect. Compared with the model control group, the apoptosis rate of cortical neurons, the content of NO and MDA in cerebral cortex, the ratio of p-JNK3/JNK3, the expression level of cytochrome C and caspase-3 protein in PNE medium-dose group were significantly reduced , and the activity of SOD, the Bcl-2/Bax ratio were significantly improved (all P<0.05). CONCLUSION: PNE ameliorates brain injury after cerebral ischemia reperfusion in rats, which may be related to scavenging NO and MDA, inhibiting oxidative stress-mediated JNK3/caspase-3 signsal transduction to inhibit neuronal apoptosis.

Realgar (As4S4), a traditional Chinese medicine, induces acute promyelocytic leukemia cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway in vitro.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myelogenous leukemia (AML) characterized by the proliferation of abnormal promyelocytes. Realgar, a Chinese medicine containing arsenic, can be taken orally. Traditional Chinese medicine physicians have employed realgar to treat APL for over a thousand years. Therefore, realgar may be a promising candidate for the treatment of APL. Nevertheless, the underlying mechanism behind realgar therapy is largely unclear. The present study aimed to investigate the effect of realgar on cell death in the APL cell line (NB4) in vitro and to elucidate the underlying mechanism. In this study, after APL cells were treated with different concentrations of realgar, the cell survival rate, apoptotic assay, morphological changes, ATP levels and cell cycle arrest were assessed. The expression of Bcl-2, Bax, Cytochrome C (Cyt-C) and apoptosis-inducing factor (AIF) at the mRNA and protein levels were also measured by immunofluorescence, quantitative PCR (qPCR) and Western blotting. We found that realgar could significantly inhibit APL cell proliferation and cell death in a time- and dose-dependent manner. Realgar effectively decreased the ATP levels in APL cells. Realgar also induced APL cell cycle arrest at the S and G2/M phases. Following realgar treatment, the mRNA and protein levels of Bcl-2 were significantly downregulated, whereas the levels of Bax, Cyt-C, and AIF were significantly upregulated. In summary, realgar can induce APL cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway, suggesting that realgar may be an effective therapeutic for APL.

APAF1-Binding Long Noncoding RNA Promotes Tumor Growth and Multidrug Resistance in Gastric Cancer by Blocking Apoptosome Assembly.

Chemotherapeutics remain the first choice for advanced gastric cancers (GCs). However, drug resistance and unavoidable severe toxicity lead to chemotherapy failure and poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in tumor progression in many cancers, including GC. Here, through RNA screening, an apoptotic protease-activating factor 1 (APAF1)-binding lncRNA (ABL) that is significantly elevated in cancerous GC tissues and an independent prognostic factor for GC patients is identified. Moreover, ABL overexpression inhibits GC cell apoptosis and promotes GC cell survival and multidrug resistance in GC xenograft and organoid models. Mechanistically, ABL directly binds to the RNA-binding protein IGF2BP1 via its KH1/2 domain, and then IGF2BP1 further recognizes the METTL3-mediated m6A modification on ABL, which maintains ABL stability. In addition, ABL can bind to the WD1/WD2 domain of APAF1, which competitively prevent cytochrome c from interacting with APAF1, blocking apoptosome assembly and caspase-9/3 activation; these events lead to resistance to cell death in GC cells. Intriguingly, targeting ABL using encapsulated liposomal siRNA can significantly enhance the sensitivity of GC cells to chemotherapy. Collectively, the results suggest that ABL can be a potential prognostic biomarker and therapeutic target in GC.

Condensed tannins from Ulmus pumila L. leaves induce G2/M phase arrest and apoptosis via caspase-cascade activation in TFK-1 cholangiocarcinoma cells.

Condensed tannins the polyphenolic compounds that are widespread in plants have been proved to have antitumor potential. Here, we purified the bioactive condensed tannins from leaves of Ulmus pumila L. and explored their structural characteristics, antitumor effect on TFK-1 cholangiocarcinoma cells as well as the related potential mechanism. The UV-Vis, FT-IR spectroscopy, ESI-Full-MS, and thiolysis-HPLC-ESI-MS demonstrated that U. pumila condensed tannins (UCTs) consisted essentially of procyanidins with epicatechin as the main flavan-3-ol extension unit. The UCTs could significantly reduce the survival rate of human cholangiocarcinoma TFK-1, SK-CHA-1, and MZ-CHA-1 cells with the better inhibitory effect on TFK-1 cell proliferation. Flow cytometric assay showed that UCTs affected TFK-1 survival by G2/M phase arrest and inducing apoptosis in a dose-dependent manner. In addition, a total of 6592 differentially expressed genes (DEGs), consisting of 94 upregulated and 6498 downregulated DEGs, were identified between untreated and UCTs-treated TFK-1 cells using RNA-seq technology. Enrichment analysis based on the KEGG database revealed that these DEGs were closely associated with cell cycle and p53 apoptotic signaling pathways. Furthermore, qRT-PCR confirmed that treatment of UCTs to TFK-1 cells caused significant changes in the expression of cyclin E, cdc25 A, cytochrome c, caspase-3, and caspase-8. These results indicated that UCTs exhibited the growth inhibition effect on TFK-1 cells possibly via G2/M cell cycle arrest and activation of caspase-cascade to induce apoptosis, and had potential as an anti-cholangiocarcinoma drug for further development. PRACTICAL APPLICATIONS: Ulmus pumila L. as a valuable tree species has been widely used in fields of medicine and food. Condensed tannins, the polyphenolic compounds widespread in plants, have been proved to have antitumor potential and be safe to normal cells. In this study, the condensed tannins from leaves of U. pumila (UCTs) remarkably suppressed cholangiocarcinoma (CCA) cell viability possibly via G2/M cell cycle arrest and activation of caspase-cascade to induce apoptosis. The results provided evidence for the application of UCTs as a potential therapeutic drug for CCA tumor.

Arylamine N-acetyltransferase 1 deficiency inhibits drug-induced cell death in breast cancer cells: switch from cytochrome C-dependent apoptosis to necroptosis.

PURPOSE: Arylamine N-acetyltransferase 1 (NAT1) deficiency has been associated with drug resistance and poor outcomes in breast cancer patients. The current study aimed to investigate drug resistance in vitro using normal breast cancer cell lines and NAT1-deficient cell lines to understand the changes induced by the lack of NAT1 that resulted in poor drug response. METHODS: The response to seven chemotherapeutic agents was quantified following NAT1 deletion using CRISPR-Cas 9 in MDA-MB-231 and T-47D cells. Apoptosis was monitored by annexin V staining and caspase 3/7 activity. Cytochrome C release and caspase 8 and 9 activities were measured by Western blots. Caspase 8 was inhibited using Z-IETD-FMK and necroptosis was inhibited using necrostatin and necrosulfonamide. RESULTS: Compared to parental cells, NAT1 depleted cells were resistant to drug treatment. This could be reversed following NAT1 rescue of the NAT1 deleted cells. Release of cytochrome C in response to treatment was decreased in the NAT1 depleted cells, suggesting suppression of the intrinsic apoptotic pathway. In addition, NAT1 knockout resulted in a decrease in caspase 8 activation. Treatment with necrosulfonamide showed that NAT1 deficient cells switched from intrinsic apoptosis to necroptosis when treated with the anti-cancer drug cisplatin. CONCLUSIONS: NAT1 deficiency can switch cell death from apoptosis to necroptosis resulting in decreased response to cytotoxic drugs. The absence of NAT1 in patient tumours may be a useful biomarker for selecting alternative treatments in a subset of breast cancer patients.

Novel Preclinical Study of Galloylquinic Acid Compounds from Copaifera lucens with Potent Antifungal Activity against Vaginal Candidiasis Induced in a Murine Model via Multitarget Modes of Action.

Vaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 µg/mL) as well as minimum fungicidal concentrations (2 to 32 µg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC50) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 µg/mL and 32 µg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1, and ALS1, were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis. IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB, and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

Potential anti-acanthamoebic effects through inhibition of CYP51 by novel quinazolinones.

Acanthamoeba spp. are free living amoebae which can give rise to Acanthamoeba keratitis and granulomatous amoebic encephalitis. The surface of Acanthamoeba contains ergosterol which is an important target for drug development against eukaryotic microorganisms. A library of ten functionally diverse quinazolinone derivatives (Q1-Q10) were synthesised to assess their activity against Acanthamoeba castellanii T4. The in-vitro effectiveness of these quinazolinones were investigated against Acanthamoeba castellanii by amoebicidal, excystation, host cell cytopathogenicity, and NADPH-cytochrome c reductase assays. Furthermore, wound healing capability was assessed at different time durations. Maximum inhibition at 50 µg/mL was recorded for compounds Q5, Q6 and Q8, while the compound Q3 did not exhibit amoebicidal effects at tested concentrations. Moreover, LDH assay was conducted to assess the cytotoxicity of quinazolinones against HaCaT cell line. The results of wound healing assay revealed that all compounds are not cytotoxic and are likely to promote wound healing at 10 µg/mL. The excystation assays revealed that these compounds significantly inhibit the morphological transformation of A. castellanii. Compound Q3, Q7 and Q8 elevated the level of NADPH-cytochrome c reductase up to five folds. Sterol 14alpha-demethylase (CYP51) a reference enzyme in ergosterol pathway was used as a potential target for anti-amoebic drugs. In this study using i-Tasser, the protein structure of Acanthamoeba castellanii (AcCYP51) was developed in comparison with Naegleria fowleri protein (NfCYP51) structure. The sequence alignment of both proteins has shown 42.72% identity. Compounds Q1-Q10 were then molecularly docked with the predicted AcCYP51. Out of ten quinazolinones, three compounds (Q3, Q7 and Q8) showed good binding activity within 3 Å of TYR 114. The in-silico study confirmed that these compounds are the inhibitor of CYP51 target site. This report presents several potential lead compounds belonging to quinazolinone derivatives for drug discovery against Acanthamoeba infections.

A nanoplatform based on mesoporous silica-coated gold nanorods for cancer triplex therapy.

To enhance the efficacy of nanoparticle-based cancer therapy with reduced side effects and promote its clinical translation, a biocompatible nanocomposite based on mesoporous silica-coated gold nanorods (AuNR@MSN) for triple tumor therapy is reported in this study. The gold core served as a hyperthermia agent, while the MSN shell acted as a reservoir of chemotherapeutics owing to its excellent loading capacity. Cytochrome c with the apoptosis inducing function was anchored on the surface of AuNR@MSN to prevent drug leakage through redox-responsive disulfide bonds. The successful construction of a nanocomposite was confirmed by characterization of the physicochemical properties. In vitro and in vivo studies demonstrated that the nanocomposite displayed an optimizing anti-tumor effect with a synergistic strategy of excellent photothermal therapy, chemotherapy and protein therapy. Therefore, this cooperative strategy paves the way for high-efficiency oncotherapy with reduced side effects.

Sequentially Site-Specific Delivery of Apoptotic Protein and Tumor-Suppressor Gene for Combination Cancer Therapy.

Nanocarrier-mediated codelivery of multiple anticancer drugs is a potential strategy for enhanced efficacy of combination cancer treatment by unifying differential pharmacokinetic properties and maintaining an optimal ratio of drug cargoes. However, a programmable codelivery system is highly desired to deliver different therapeutics to their specific sites of action to pursue maximized combinational effect. Herein a liposome-based nanoassembly (p53/C-rNC/L-FA) is developed for intracellular site-specific delivery of an apoptotic protein cytochrome c (CytoC) and a plasmid DNA encoding tumor-suppressing p53 protein (p53 DNA). p53/C-rNC/L-FA consists of an acid-activated fusogenic liposomal membrane shell modified with folic acid (L-FA) and a DNA/protein complex core assembled by the p53 DNA, protamine and CytoC-encapsulated redox-responsive nanocapsule (C-rNC). Intratumoral and intraendosomal acidities promote membrane fusion between liposome and biomembrane, resulting in release of the encapsulated p53/C-rNC complex into the cytoplasm. The cytoplasmic reduction causes degradation of C-rNC with release of CytoC that induces tumor cell apoptosis. The p53 DNA is transported into the nucleus by the aid of the cationic protamine and thus generates expression of the p53 protein that enhances apoptosis combined with CytoC. p53/C-rNC/L-FA is demonstrated to significantly induce tumor cell apoptosis and inhibit tumor growth in the orthotopic breast tumor mouse model.

Inducing cell death in vitro in cancer cells by targeted delivery of cytochrome c via a transferrin conjugate.

One of the major drawbacks of many of the currently used cancer drugs are off-target effects. Targeted delivery is one method to minimize such unwanted and detrimental events. To actively target lung cancer cells, we have developed a conjugate of the apoptosis inducing protein cytochrome c with transferrin because the transferrin receptor is overexpressed by many rapidly dividing cancer cells. Cytochrome c and transferrin were cross-linked with a redox sensitive disulfide bond for the intra-cellular release of the protein upon endocytosis by the transferrin receptor. Confocal results demonstrated the cellular uptake of the cytochrome c-transferrin conjugate by transferrin receptor overexpressing A549 lung cancer cells. Localization studies further validated that this conjugate escaped the endosome. Additionally, an in vitro assay showed that the conjugate could induce apoptosis by activating caspase-3. The neo-conjugate not only maintained an IC50 value similar to the well known drug cisplatin (50 µM) in A549 cancer cells but also was nontoxic to the normal lung (MRC5) cells. Our neo-conjugate holds promise for future development to target cancers with enhanced transferrin receptor expression.

The fabrication of double-layered chitosan/gelatin/genipin nanosphere coating for sequential and controlled release of therapeutic proteins.

Bone regeneration is a complicated process and includes a number of distinct and sequential stages of coordinated cellular actions under the regulation of multiple growth factors. Therefore, bone grafting materials in which growth factors can be incorporated and released in a programmed order in line with the bone tissue healing process may lead to desirable clinical outcomes. In the present study, a double-layered chitosan/gelatin/genipin (d-CSG/G) nanosphere coating is developed by using layer-by-layer electrophoretic deposition and genipin crosslinking. The surface morphology, physicochemical and mechanical properties of the coatings are explored. Cytochrome C is used as a therapeutic model protein and is successfully loaded on the inner and outer layers of the coating. The protein release can be controlled by the loading position, genipin concentration and thickness of the outer layer. Furthermore, the cell response to the coatings was evaluated. Real-time polymerase chain reactions, immunofluorescence staining and extracellular matrix mineralization assay confirmed that the functions of the loaded growth factor are fully preserved after fabrication. Overall, the d-CSG/G nanosphere coating could be a promising growth factor delivery system to promote bone tissue regeneration.

Multi-stage, charge conversional, stimuli-responsive nanogels for therapeutic protein delivery.

A boronate ester crosslinked zwitterionic nanogel (NGCA) with ATP/pH-sensitivity has been developed with an inverse nanoprecipitation technique to achieve a two-stage charge conversion that responds to tumor extracellular conditions (pH 6.5-6.8) and an intracellular acidic environment (pH 5-6). Cationic cytochrome C (CC), a therapeutic protein, has been encapsulated into NGCA through inverse nanoprecipitation via electrostatic interactions to form protein-loaded nanogel (NGCA-CC). By adjusting the ratio of the amino and carboxyl groups in the nanogels, negatively charged nanogels that are safer under physiological conditions (pH 7.4) can convert their surface charge to positive at tumor extracellular pH, which enhance their cellular uptake efficiency. The citraconic amide formed from citraconic anhydride and amine can be cleaved in the intracellular acidic organelles to expose more amino groups and facilitate endosomal escape. The release of CC is accelerated in the presence of 5 mM ATP or under acidic conditions. Confocal laser scanning microscopy (CLSM) and flow cytometry have shown that NGCA-CC's cell uptake is higher at pH 6.5 than at pH 7.4. MTT and real-time cell analysis (RTCA) have illustrated that there is more toxicity at pH 6.5 than at pH 7.4. The apoptosis process induced by CC was determined by flow cytometry.

The study of the complexes of nitromedicine with cytochrome c and NO-containing aqueous dosage form in the wound treatment of rats.

The interaction of cytochrome c with nitromedicines, such as 5-nitrofural, 5-nitroxoline, metronidazole and sodium nitrite which enables the generation of nitric oxide or nitrosyl complexes in the presence of ascorbic acid or sodium ascorbate in acid medium has been investigated. The pharmaceutical compositions containing cytochrome c and nitromedicine complexes as active substances were studied in the experiments by using rats. It has been shown that positive local and systemic effects were estimated when NO-containing gel was used at burn treatment. These positive effects at the local level are due to a sufficient microcirculation index which indicates intensification of the blood flow in the microvessels in the injured area. These effects at the systemic level provide maintenance of the general heart rhythm and gradual recovery of the vegetative balance which is not observed in the animals of the control group.

Growth inhibition and apoptosis-inducing effect on human cancer cells by RCE-4, a spirostanol saponin derivative from natural medicines.

Reineckia carnea has been used to treat several diseases in folk remedies. RCE-4 has been isolated from several plants of the family Liliaceae, but its biological activity has not yet been reported. In the present study, we found that RCE-4 exhibited potent cytotoxicity to the tested human cancer cell lines, and the CaSki cell line was the most sensitive with an IC50 of 3.37 µM. Thus, we presented the apoptosis-inducing effect of RCE-4 on CaSki cervical cancer cells and investigated the relevant mechanisms. Based on observations using transmission electron microscopy, RCE-4-treated cells manifested nuclear shrinkage, condensation and fragmentation. Annexin V/PI dual staining flow cytometry assay further confirmed that RCE-4 caused a dose-dependent early apoptotic effect. Prior to these events, RCE-4 triggered a rapid decrease of the mitochondrial membrane potential and caused the release of cytochrome c from the mitochondria into the cytoplasm. RCE-4 increased the expression of Bax and decreased the expression of Bcl-2, thus augmenting the Bax/Bcl-2 ratio. These findings suggest that RCE-4 induces mitochondrial-mediated apoptosis in CaSki cells and has the potential to be developed as an anticancer agent.

The targeted intracellular delivery of cytochrome C protein to tumors using lipid-apolipoprotein nanoparticles.

Intracellular-acting therapeutic proteins offer a promising clinical alternative to extracellular-acting agents, but are limited in efficacy by their low permeability into the cell cytoplasm. We have developed a nanoparticle (NP) composed of lipid (DOTAP/DOPE) and apolipoprotein (APOA-I) to mediate the targeted delivery of intracellular-acting protein drugs to non-small cell lung tumors. NPs were produced with either GFP, a fluorescent model protein, or cytochrome C (cytC), an inducer of apoptosis in cancer cells. GFP and cytC were separately conjugated with a membrane permeable sequence (MPS) peptide and were admixed with DOPE/DOTAP nanoparticle formulations to enable successful protein loading. Protein-loaded NPs were modified with DSPE-PEG-Anisamide to enable specific NP targeting to the tumor site in a xenograft model. The resulting particle was 20-30 nm in size and exhibited a 64-75% loading efficiency. H460 cells treated with the PEGylated MPS-cytC-NPs exhibited massive apoptosis. When MPS-GFP-NPs or MPS-cytC-NPs were intravenously administered in H460 tumor bearing mice, a specific tumor targeting effect with low NP accumulation in the liver was observed. In addition, MPS-cytC-NP treatment provoked a tumor growth retardation effect in H460 xenograft mice. We conclude that our NP enables targeted, efficacious therapeutic protein delivery for the treatment of lung cancer.

[Hepatoprotective properties of cytochrome C in traumatic toxicosis].

Experiments on rats showed that traumatic toxicosis (crush syndrome) was accompanied by disorders of both excretion and detoxication functions of the liver and a decrease in the energy potential of the liver. Systemic administration of cytochrome C (10 mg/kg) immediately after trauma and decompression increased the level of endogenous cytochrome C, recovered the pool of adenine nucleotides, normalized bromsulfaleine excretion from the blood, and decreased the content of toxic metabolites in the blood. The obtained experimental data show that cytochrome C possesses high hepatoprotective properties with respect to the development of traumatic toxicosis.