Mitochondrial Membrane Damage Research Articles

Mitochondrial administration alleviates lead- and cadmium-induced toxicity in rat renal cells.

The role of heavy metals such as lead (Pb) and cadmium (Cd) in the etiology of many diseases has been proven. Also, these heavy metals can affect the normal mitochondrial function. Mitochondrial administration therapy is one of the methods used by researchers to help improve mitochondrial defects and diseases. The use of isolated mitochondria as a therapeutic approach has been investigated in in vivo and in vitro studies. Accordingly, in this study, the effects of mitochondrial administration on the improvement of toxicity caused by Pb and Cd in renal proximal tubular cells (RPTC) have been investigated. The results showed that treatment to Pb and Cd caused an increase in the level of free radicals, lipid peroxidation (LPO) content, mitochondrial and lysosomal membrane damage, and also a decrease in the reduced glutathione content in RPTC. In addition, reports have shown an increase in oxidized glutathione content and changes in energy (ATP) levels. Following, the results have shown the protective role of mitochondrial administration in improving the toxicity caused by Pb and Cd in RPTC. Furthermore, the mitochondrial internalization into RPT cells is mediated through actin-dependent endocytosis. So, it could be suggested that the treatment of Pb- and Cd-induced cytotoxicity in RPTC could be carried out through mitochondria administration.

Ceramides from sea red rice bran ameliorate oxidative stress and lifespan in Caenorhabditis elegans by activating the p38/MAPK signaling pathway and regulating the gut microbiota

Sea red rice bran, a new salinity-tolerant sea rice by-product, contains high levels of ceramides (Cers), which are necessary to tap into their bioactivity for improving the health effects of the organism. This study aimed to investigate the effect of Cers on the improving the oxidative stress and lifespan of C. elegans, and the relationship of the interaction through the p38/MAPK signaling pathway and under the regulation of intestinal flora. The results showed that Cers treatment effectively reversed the oxidative damage caused by paraquat and hydrogen peroxide in C. elegans and reduced overexpression of ROS and accumulation of mitochondrial membrane damage. In addition, Cers intake successfully activated all gene targets of p38/MAPK signaling pathway, significantly promoted the migration of SKN-1 to the nucleus, and increased the abundance of probiotic bacteria such as Ruminoccus, AKKermansia, and Lactobacillus in the intestinal flora and effectively mitigated the adverse effects of oxidative stress. Therefore, these results indicate that Cers could be effectively in ameliorating the level of oxidative stress in the body, thereby increasing longevity and health indicators. Providing a theoretical basis for the prevention of aging-related chronic diseases and health care, and enhancing the high-value comprehensive utilization of sea red rice by-products.

BMSC-Derived Exosomes Attenuate Rat Osteoarthritis by Regulating Macrophage Polarization through PINK1/Parkin Signaling Pathway.

Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) may modulate the M1/M2 polarization of macrophages during osteoarthritis (OA). However, the underlying mechanisms of BMSC-Exos in this process still need to be elucidated. In this study, we explored the role of BMSC-Exos in the polarization of macrophages in vitro and the OA rats in vivo. The effects of BMSC-Exos on RAW264.7 cells were determined, including the production of reactive oxygen species (ROS) and the protein expression of Akt, PINK1, and Parkin. We prepared an OA model by resecting the anterior cruciate ligament and medial meniscus of Sprague-Dawley (SD) rats. Hematoxylin-eosin (H&E) and safranin O-fast green staining, immunohistochemistry and immunofluorescence analyses, and the examination of interleukin 6 (IL-6), interleukin 1β (IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin 10 (IL-10) were performed to assess changes in cartilage and synovium. BMSC-Exos inhibited mitochondrial membrane damage, ROS production, and the protein expression of PINK1 and Parkin. Akt phosphorylation was downregulated under lipopolysaccharide (LPS) induction but significantly recovered after treatment with BMSC-Exos. BMSC-Exos alleviated cartilage damage, inhibited M1 polarization, and promoted M2 polarization in the synovium in OA rats. The expression of PINK1 and Parkin in the synovium and the levels of IL-6, IL-1β, and TNF-α in the serum decreased, but the level of IL-10 increased when BMSC-Exos were used in OA rats. BMSC-Exos ameliorate OA development by regulating synovial macrophage polarization, and one of the underlying mechanisms may be through inhibiting PINK1/Parkin signaling.

Mitochondrial Transplantation Alleviates Doxorubicin-Induced Toxicity in Rat Renal Cells

Background: Doxorubicin (DOX) is used in the treatment of various cancers and has good effectiveness. However, its therapeutic use is limited due to its effects on various organs and healthy cells. Doxorubicin can affect the kidneys and cause toxicity. Evidence shows that DOX induces nephrotoxicity through oxidative stress. Objectives: In this research, we examined the effect of mitochondrial transplantation on improving mitochondrial and cellular toxicity caused by DOX on renal proximal tubular cells (RPTCs). Methods: The research measured 7 toxicity parameters, including cell lysis, reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) decline, GSH and GSSG content, lipid peroxidation (LPO), adenosine triphosphate (ATP) content, and Caspase-3 activity (the final mediator of apoptosis). Active fresh mitochondria were prepared from Wistar rat kidney. Results: The findings indicated that DOX caused cytotoxicity in RPTCs. Additionally, DOX induced oxidative stress by increasing the level of reactive oxygen species, reducing glutathione content, and elevating lipid peroxidation. Moreover, it led to damage to the mitochondrial membrane, increased caspase-3 activity, and decreased ATP content. Mitochondrial transplantation, as a new therapeutic approach, reduced oxidative stress, mitochondrial membrane damage, and apoptosis caused by DOX in RPTCs. Furthermore, this therapeutic approach increased the ATP content in RPTCs. Conclusions: Our study suggests that this therapeutic approach could be helpful in the treatment of drug-induced nephrotoxicity.

A genetically encoded fluorescent protein sensor for mitochondrial membrane damage detection

Mitochondria are essential cellular organelles; detecting mitochondrial damage is crucial in cellular biology and toxicology. Compared with existing chemical probe detection methods, genetically encoded fluorescent protein sensors can directly indicate cellular and molecular events without involving exogenous reagents. In this study, we introduced a molecular sensor system, MMD-Sensor, for monitoring mitochondrial membrane damage. The sensor consists of two molecular modules. Module I is a fusion structure of the mitochondrial localization sequence (MLS), AIF cleavage site sequence (CSS), nuclear localization sequence (NLS), N-terminus of mNeonGreen and mCherry. Module II is a fusion structure of the C-terminus of mNeonGreen, NLS sequence, and mtagBFP2. Under normal condition, Module I is constrained in the inner mitochondrial membrane anchored by MLS, while Module II is restricted to the nucleus by its NLS fusion component. If the mitochondrial membrane is damaged, CSS is cut from the inner membrane, causing Module I to shift into the nucleus guided by the NLS fusion component. After Module I enters the nucleus, the N- and C-terminus of mNeonGreen meet each other and rebuild its intact 3D structure through fragment complementation and thus generates green fluorescence in the nucleus. Dynamic migration of red fluorescence from mitochondria to the nucleus and generation of green fluorescence in the nucleus indicate mitochondrial membrane damage. Using the MMD-Sensor, mitochondrial membrane damage induced by various reagents, such as uncoupling agents, ATP synthase inhibitors, monovalent cationic carriers, and ROS, in HeLa and 293T cells are directly observed and evaluated.

Microcirculation-driven mitochondrion dysfunction during the progression of experimental sepsis

Sepsis is accompanied by a less-known mismatch between hemodynamics and mitochondrial respiration. We aimed to characterize the relationship and time dependency of microcirculatory and mitochondrial functions in a rodent model of intraabdominal sepsis. Fecal peritonitis was induced in rats, and multi-organ failure (MOF) was evaluated 12, 16, 20, 24 or 28 h later (n = 8/group, each) using rat-specific organ failure assessment (ROFA) scores. Ileal microcirculation (proportion of perfused microvessels (PPV), microvascular flow index (MFI) and heterogeneity index (HI)) was monitored by intravital video microscopy, and mitochondrial respiration (OxPhos) and outer membrane (mtOM) damage were measured with high-resolution respirometry. MOF progression was evidenced by increased ROFA scores; microcirculatory parameters followed a parallel time course from the 16th to 28th h. Mitochondrial dysfunction commenced with a 4-h time lag with signs of mtOM damage, which correlated significantly with PPV, while no correlation was found between HI and OxPhos. High diagnostic value was demonstrated for PPV, mtOM damage and lactate levels for predicting MOF. Our findings indicate insufficient splanchnic microcirculation to be a possible predictor for MOF that develops before the start of mitochondrial dysfunction. The adequate subcellular compensatory capacity suggests the presence of mitochondrial subpopulations with differing sensitivity to septic insults.

Phytochemical profile by LC-MS/MS analysis and evaluation of antioxidant, antidiabetic, anti-Alzheimer, and anticancer activity of Onobrychis argyrea leaf extracts

IntroductionStudies on different species of Onobrychis, a member of the Fabaceae family, have revealed a number of biological activities with potential applications in alternative medicine. The phytochemical content of the plant Onobrychis argyrea subsp. argyrea and some of the important biological activities linked to its metabolism are quite limited. Therefore, this study was the first to determine the content of secondary metabolites in this species and to elucidate important biological activities related to metabolism. MethodsLC-MS/MS was used for the quantitative analysis of bioactive compounds in the aerial part of O. argyrea extracts. The antioxidant properties of the extracts were assessed based on their ability to scavenge DPPH radicals and reduce iron ions. The anti-diabetic effect of the extracts was tested by the inhibition of α-amylase and α-glycosidase enzymes, and the anti-Alzheimer's ability was tested by the inhibition of the enzymes AChE and BChE. The cytotoxic effects of the extracts were tested on four different cancer lines, namely A-549, HT-29, MCF-7 and MDA-MB 453 using the XTT assay. The mechanism of the anti-cancer effect was determined by means of flow cytometry analysis on HT-29 cells. ResultsTwenty-nine phytochemicals were identified via the LC-MS/MS analysis in the plant extracts. The most abundant phytochemicals in the extracts were quinic acid, isoquercitrin, epicatechin, and routine, respectively. Antioxidant analysis showed that among all extracts, methanol extract (ME) was most effective in scavenging DPPH (IC50: 23.93 ± 0.96 µg/mL) and in reducing iron ions (53.24 ± 2.14 mgTEAC/g). Enzyme activity studies demonstrated that ME is a potent inhibitor of α-glycosidase (IC50: 15.06±0.64 µg/mL), AChE (IC50: 24.75±0.76 µg/mL), and BChE (IC50: 3.36±0.14 µg/mL). XTT assay results disclosed the strongest anti-proliferative activity of ME against the human colorectal adenocarcinoma HT-29 cell line with an IC50 value of 33.35±0.92 µg/mL. Flow cytometry analysis revealed that ME caused mitochondrial membrane damage in HT-29 cells (**p<0.01) and subsequently induced apoptosis (**p<0.01) by activating the caspase pathway (**p<0.001). ConclusionO. argyrea, rich in bioactive compounds, showed remarkable antioxidant, antidiabetic, Alzheimer's, and anticancer properties in vitro, demonstrating that this plant contains secondary metabolites that may be potential drug candidates for the treatment of metabolic diseases.

Nickel-doped vanadium pentoxide (Ni@V2O5) nanocomposite induces apoptosis targeting PI3K/AKT/mTOR signaling pathway in skin cancer: An in vitro and in vivo study

In the present study, the vanadium pentoxide (V2O5) nickel-doped vanadium pentoxide (Ni@V2O5) was prepared and determined for in vitro anticancer activity. The structural characterization of the prepared V2O5 and Ni@V2O5 was determined using diverse morphological and spectroscopic analyses. The DRS-UV analysis displayed the absorbance at 215 nm for V2O5 and 331 nm for Ni@V2O5 as the primary validation of the synthesis of V2O5 and Ni@V2O5. The EDS spectra exhibited the presence of 30% of O, 69% of V, and 1% of Ni and the EDS mapping showed the constant dispersion. The FE-SEM and FE-TEM analysis showed the V2O5 nanoparticles are rectangle-shaped and nanocomposites have excellent interfaces between nickel and V2O5. The X-ray photoelectron spectroscopy (XPS) investigation of Ni@V2O5 nanocomposite endorses the occurrence of elements V, O, and Ni. The in vitro MTT assay clearly showed that the V2O5 and Ni@V2O5 have significantly inhibited the proliferation of B16F10 skin cancer cells. In addition, the nanocomposite produces the endogenous reactive oxygen species in the mitochondria, causes the mitochondrial membrane and nuclear damage, and consequently induces apoptosis by caspase 9/3 enzymatic activity in skin cancer cells. Also, the western blot analysis showed that the nanocomposite suppresses the oncogenic marker proteins such as PI3K, Akt, and mTOR in the skin cancer cells. Together, the results showed that Ni@V2O5 can be used as an auspicious anticancer agent against skin cancer.

Role of Oxidative Stress and Reactive Metabolites in Cytotoxicity & Mitotoxicity of Clozapine, Diclofenac and Nifedipine in CHO-K1 Cells In Vitro.

To provide in vitro data on toxicity mechanisms of clozapine, diclofenac and nifedipine. CHO-K1 cells were used as in vitro model to explore mechanisms of cytotoxicity of the test drugs. Cytotoxic mechanisms of clozapine (CLZ), diclofenac (DIC) and nifedipine (NIF) were studied in CHO-K1 cells in vitro. All three drugs induce adverse reactions in some patients with partially unknown mechanisms. Following the determination of time- and dose-dependency of cytotoxicity by the MTT test, cytoplasmic membrane integrity was explored by the LDH leakage test. Both end-points were further examined in the presence of soft and hard nucleophilic agents, glutathione (GSH) and potassium cyanide (KCN), respectively, and either individual or general cytochrome P450 (CYP) inhibitors, whether CYP-catalysed formation of electrophilic metabolites play a role in the observed cytotoxicity and membrane damage. The generation of reactive metabolites during the incubations was also explored. Formation of malondialdehyde (MDA) and oxidation of dihydrofluorescein (DCFH) were monitored whether peroxidative membrane damage and oxidative stress take place in cytotoxicity. Incubations were also conducted in the presence of chelating agents of EDTA or DTPA to explore any possible role of metals in cytotoxicity by facilitating electron transfer in redox reactions. Finally, mitochondrial membrane oxidative degradation and permeability transition pore (mPTP) induction by the drugs were tested as markers of mitochondrial damage. The presence of an individual or combined nucleophilic agents significantly diminished CLZ- and NIF-induced cytotoxicities, while the presence of both agents paradoxically increased DIC-induced cytotoxicity by a factor of three with the reason remaining unknown. The presence of GSH significantly increased DIC-induced membrane damage too. Prevention of membrane damage by the hard nucleophile KCN suggests the generation of a hard electrophile upon DIC and GSH interaction. The presence of CYP2C9 inhibitor sulfaphenazol significantly diminished DIC-induced cytotoxicity, probably by preventing the formation of 4-hydroxylated metabolite of DIC, which further converts to an electrophilic reactive intermediate. Among the chelating agents, EDTA caused a marginal decrease in CLZ-induced cytotoxicity, while DIC-induced cytotoxicity was amplified by a factor of five. Both reactive and stable metabolites of CLZ could be detected in the incubation medium of CLZ with CHO-K1 cells, which are known to have low metabolic capacity. All three drugs caused a significant increase in cytoplasmic oxidative stress by means of DCFH oxidation, which was confirmed by increased MDA from cytoplasmic as well as mitochondrial membranes. The addition of GSH paradoxically and significantly increased DIC-induced MDA formation, in parallel with the increase in membrane damage when DIC and GSH combined. Our results suggested that the soft electrophilic nitrenium ion of CLZ is not responsible for the observed in vitro toxicities, and this may originate from a relatively low amount of the metabolite due to the low metabolic capacity of CHO-K1. A hard electrophilic intermediate may contribute to cellular membrane damage incubated with DIC, while a soft electrophilic intermediate seems to exacerbate cell death by a mechanism other than membrane damage. A significant decrease in cytotoxicity of NIF by GSH and KCN suggested that both soft and hard electrophiles contribute to NIF-induced cytotoxicity. All three drugs induced peroxidative cytoplasmic membrane damage, while only DIC and NIF induced peroxidative mitochondrial membrane damage, which suggested mitochondrial processes may contribute to adverse effects of these drugs in vivo.

Polysorbate 80 surface modified SLNs of formoterol suppress SNCA gene and mitochondrial oxidative stress in mice model of Parkinson’s disease

The present study hypothesises that the selective brain β2 receptor activation through β2-adrenoreceptor agonist (β2ARA), Formoterol (FMT), suppresses SNCA gene expression, a pathological hallmark of Parkinson’s disease (PD) in brain. Further, it is also hypothesized that brain targeted delivery of Formoterol via polysorbate-80 surface modified solid lipid nanoparticles of Formoterol (FMT-SLNs-PS80) can improve its stability, therapeutic efficacy and avoid/reduce peripheral off-target side effects. FMT-SLNs-PS80 was prepared by solvent injection method, the formulation was optimized by using Box–Behnken design and characterized by measuring drug content, entrapment efficacy, particle size, zeta potentials and poly dispersibility. The FMT-SLNs-PS80, significantly decreases the SNCA expression, mitochondrial membrane damage and rotenone induced changes in oxidative (SOD, CAT, GSH and ROS) stress markers in SH-SY5Y cell lines. The ex vivo permeation study of the formulation using everted chicken ileum exhibited a steady state flux. The pharmacokinetic and tissue distribution studies of the formulation in rats showed a significant improvement in the kinetic parameters when compared to naïve FMT, further the formulation also improved the brain bioavailability of FMT. The anti-Parkinson’s efficacy studies of the formulation in mice showed a significant neuroprotection against rotenone-induced changes in behavioural and biochemical parameters. Further, the histopathological analysis of mice brain confirms a significant neuroprotective benefit. The present study successfully establishes the brain targeted delivery and anti-Parkinson’s therapeutic efficacy of FMT-SLNs-PS80.

Combinatorial anticancer effects of multi metal ion and drug substitute with hydroxyapatite coatings on surgical grade 316LSS stainless steel alloys towards biomedical applications

The present study highlights the metal ions and existing drug ampicillin-loaded nano-hydroxyapatite (nHAP) coated on 316 stainless steel alloy (316LSS) using the electrodeposition method. In addition, the sample phase purity and crystallinity of the HAp composite coatings on 316LSS were analyzed by X-ray diffraction analysis (XRD) and Fourier transform infrared (FT-IR) spectroscopy using the KBr pellet method. The coating microstructure/morphology was characterized using a scanning electron microscope, which showed a flower-like morphology with the attachment of energy-dispersive X-ray spectroscopy (EDX) to confirm the major element groups in the synthesized sample. The TGA results showed that weight loss occurred in the temperature range of up to 800 °C for oxidation and reduction reactions. Additionally, the beat-optimized nanomaterial showed promising in vitro bioactivity against disease-causing microorganisms, displaying a significant zone of inhibition. Furthermore, cytotoxicity assessment using the MTT assay demonstrated that the synthesized samples effectively inhibited cell proliferation in human osteosarcoma cell lines (Saos-2). AO/EB double staining performed under a fluorescence microscope indicated changes in the morphology of the apoptotic cell nuclei. In addition, the relationship between ROS and nanomaterial-induced apoptosis in Saos-2 cells was analyzed by flow cytometry. MMP (ΔѰmt) profiling showed that the HAp composite increased the effective mitochondrial membrane damage or depolarization in the human osteosarcoma cell line. Moreover, the TUNEL assay suggested that treated nanomaterial with Saos-2 cells showed enhanced green fluorescence intensity, indicating terminal DNA damage. The obtained results meet the recommended physiological standard for synthesized 316LSS HAp composites to guide bone tissue regeneration.

Novel Pyrazole-Based Benzofuran Derivatives as Anticancer Agents: Synthesis, Biological Evaluation, and Molecular Docking Investigations.

In this work, the design, synthesis, and mechanistic studies of novel pyrazole-based benzofuran derivatives 1-8 as anticancer agents were discussed. Cytotoxic potency of the title compounds was evaluated against the lung carcinoma A-549, human-derived colorectal adenocarcinoma HT-29, breast adenocarcinoma MCF-7 cells as well as mouse fibroblast 3T3-L1 cells using XTT assay. Anticancer mechanistic studies were carried out with flow cytometry. XTT results revealed that all compounds exhibited dose-dependent anti-proliferative activity against the tested cancer cells, and especially compound 2 showed the strongest anti-proliferative activity with an IC50 value of 7.31 μM and the highest selectivity (15.74) on MCF-7 cells. Flow cytometry results confirmed that the cytotoxic power of compound 2 on MCF-7 cells is closely related to mitochondrial membrane damage, caspase activation, and apoptosis orientation. Finally, molecular docking studies were applied to determine the interactions between compound 2 and caspase-3 via in-silico approaches. By molecular docking studies, free binding energy (ΔGBind), docking score, Glide score values as well as amino acid residues in the active binding site were determined. Consequently, these results constitute preliminary data for in vivo anticancer studies and have the potential as a chemotherapeutic agent.

Bovine sperm HSP-70 molecules: a potential cryo-tolerance marker associated with semen quality and fertility rate.

Freezability is the ability of sperm to maintain its vitality and quality from various stress during the cryopreservation process, which is very important for the success of fertilization in AI programs. Heat shock proteins (HSPs) are unique proteins induced in response to various stress, including excess reactive oxygen species (ROS) and oxidative damage to intracellular enzymes that can harm cells. This study aimed to analyze the potential of HSP-70 molecules in bovine sperm as a marker of freezability or cryo-tolerance, as well as its association with semen quality and fertility rate. The classification of bulls is based on freezability (good freezability/GF and poor freezability/PF), which is obtained from the value of post-thaw viability using the SYBR-14/PI-flow cytometry. Semen quality assessed included sperm motility and kinetics (computer-assisted sperm analyses), plasma membrane integrity (HOS test), acrosome integrity (FITC-PNA), mitochondrial membrane (JC-1), and DNA damage (Halomax kit). The bull fertility rate assessment was analyzed based on the first service conception rate of each bull derived from data on the success of artificial insemination contained in the Indonesian-integrated National Animal Health Information System (iSIKHNAS). Gene expression levels of HSP-70 bovine sperm were performed using the RT-qPCR method. The protein abundance of HSP-70 bovine sperm was determined using the enzyme immunoassay (EIA) method. Bovine sperm HSP-70 molecules, at the gene and protein level, showed a higher abundance in GF (p < 0.05) than in PF bulls. The percentage of each parameter of frozen-thawed sperm quality was significantly higher in GF (p < 0.05) than in PF bulls. The HSP-70 molecules at the gene and protein levels were significantly positively correlated (p < 0.01) with the fertility rate. Furthermore, HSP-70 molecules were negatively associated (p < 0.01) with low mitochondrial membrane potential and sperm DNA damage and positively correlated (p < 0.01) with other frozen-thawed sperm quality parameters. The overall quality of frozen-thawed sperm was closely related (p < 0.01) to the fertility rate. We may conclude that HSP-70 molecules in bovine sperm at the gene and protein level have the potential to be developed as a marker for cryo-tolerance or freezability, which may be utilized as a predictor of fertility and frozen-thawed sperm quality in bulls.

SCP2 mediates the transport of lipid hydroperoxides to mitochondria in chondrocyte ferroptosis

Sterol carrier protein 2 (SCP2) is highly expressed in human osteoarthritis (OA) cartilage, accompanied by ferroptosis hallmarks, especially the accumulation of lipid hydroperoxides (LPO). However, the role of SCP2 in chondrocyte ferroptosis remains unexplored. Here, we identify that SCP2 transports cytoplasmic LPO to mitochondria in RSL3-induced chondrocyte ferroptosis, resulting in mitochondrial membrane damage and release of reactive oxygen species (ROS). The localization of SCP2 on mitochondria is associated with mitochondrial membrane potential, but independent of microtubules transport or voltage-dependent anion channel. Moreover, SCP2 promotes lysosomal LPO increase and lysosomal membrane damage through elevating ROS. However, SCP2 is not directly involved in the cell membrane rupture caused by RSL3. Inhibition of SCP2 markedly protects mitochondria and reduces LPO levels, attenuating chondrocyte ferroptosis in vitro and alleviating the progression of OA in rats. Our study demonstrates that SCP2 mediates the transport of cytoplasmic LPO to mitochondria and the spread of intracellular LPO, accelerating chondrocyte ferroptosis.

Therapeutic efficacy of sorafenib and plant-derived phytochemicals in human colorectal cancer cells

BackgroundThe present study aimed to investigate the sequence-dependent anticancer effects of combined treatment with sorafenib (Sora), a Food and Drug Administration-approved multikinase inhibitor drug, and plant-derived phytochemicals (PPCs) on human colorectal cancer (CRC) cell growth, and proteins associated with the control of cell cycle and apoptosis.MethodsThe cytotoxic effects of 14 PPCs on CRL1554 fibroblast cells were determined using an MTT assay. Moreover, the cytotoxicity of Sora, PPCs, and a combination of both on CRC cells were also investigated. Cell cycle analysis was performed using flow cytometry, and cell apoptosis was investigated using DNA fragmentation, Annexin V/propidium iodide double staining, and mitochondrial membrane potential analyses. The cell cycle- and apoptosis-associated protein expression levels were analysed using western blotting.ResultsBased on their low levels of cytotoxicity in CRL1554 cells at ≤ 20%, curcumin, quercetin, kaempferol, and resveratrol were selected for use in subsequent experiments. The combined treatment of sora and PPCs caused levels of CRC cytotoxicity in a dose-, cell type-, and schedule-dependent manner. Moreover, the combined treatment of CRC cells arrested cell growth at the S and G2/M phases, induced apoptotic cell death, caused extensive mitochondrial membrane damage, and altered the expression of the cell cycle and apoptotic proteins.ConclusionsResults of the present study highlighted a difference in the level of sora efficacy in CRC cells when combined with PPCs. Further in vivo and clinical studies using the combined treatment of sora and PPCs are required to determine their potential as a novel therapeutic strategy for CRCs.

New insights into the anti-apoptotic mechanism of natural polyphenols in complex with Bax protein

Excessive apoptosis can kill normal cells and lead to liver damage, heart failure and neurodegenerative diseases. Polyphenols are secondary metabolites of plants that can interact with proteins to inhibit toxins and disease-related apoptosis. Bax is the major pro-apoptotic protein that disrupts the outer mitochondrial membrane to induce apoptosis, but limited studies have focused on the interaction between polyphenols and Bax and the associated anti-apoptotic mechanisms, especially at the atomic level. In this article, we collected 69 common polyphenols for active ingredient screening targeting Bax. Polyphenols with better and worse molecular docking scores were selected, and their anti-apoptosis effects were compared using the H2O2-induced HepG2 cell model. The interactions between the selected polyphenols and Bax protein were analyzed using molecular dynamics simulation to explore the molecular mechanism underlying the anti-apoptosis effect. Secoisolariciresinol diglucoside (SDG) and Epigallocatechin-3-gallate (EGCG) with the best affinity for Bax (−6.76 and −6.52 kcal/mol) reduced the expression of cytochrome c and caspase 3, decreasing the apoptosis rate from 52 to 11% and 12%. Molecular dynamics simulation results showed that Bim unfolded the α1-α2 loop of Bax, and disrupted the non-bond interactions between the loop (Pro-43, Glu-44 and Leu-45) and surface (Ile-133, Arg-134 and Met-137) residues, with binding free energy changed from −15.0 to 0 kJ/mol. The hydrogen bonds and van der Waals interactions formed between polyphenols and Bax prevented the unfolding of the loop. Taken together, our results proved that polyphenols can inhibit apoptosis by maintaining the unactivated conformation of Bax to reduce outer mitochondrial membrane damage. Communicated by Ramaswamy H. Sarma

Alpha pyrrolidinovalerophenone (α-PVP) administration impairs spatial learning and memory in rats through brain mitochondrial dysfunction

Novel psychoactive substances (NPS) consumption has increased in recent years, thus NPS-induced cognitive decline is a current source of concern. Alpha-pyrrolidinovalerophenone (α-PVP), as a member of NPS, is consumed throughout regions like Washington, D.C., Eastern Europe, and Central Asia. Mitochondrial dysfunction plays an essential role in NPS-induced cognitive impairment. Meanwhile, no investigations have been conducted regarding the α-PVP impact on spatial learning/memory and associated mechanisms. Consequently, our study investigated the α-PVP effect on spatial learning/memory and brain mitochondrial function. Wistar rats received different α-PVP doses (5, 10, and 20 mg/kg) intraperitoneally for 10 sequential days; 24 h after the last dose, spatial learning/memory was evaluated by the Morris Water Maze (MWM). Furthermore, brain mitochondrial protein yield and mitochondrial function variables (Mitochondrial swelling, succinate dehydrogenase (SDH) activity, lipid peroxidation, Mitochondrial Membrane Potential (MMP), Reactive oxygen species (ROS) level, brain ADP/ATP proportion, cytochrome c release, Mitochondrial Outer Membrane (MOM) damage) were examined. α-PVP higher dose (20 mg/kg) significantly impaired spatial learning/memory, mitochondrial protein yield, and brain mitochondrial function (caused reduced SDH activity, increased mitochondrial swelling, elevated ROS generation, increased lipid peroxidation, collapsed MMP, increased cytochrome c release, elevated brain ADP/ATP proportion, and MOM damage). Moreover, the lower dose of α-PVP (5 mg/kg) did not alter spatial learning/memory and brain mitochondrial function. These findings provide the first evidence regarding impaired spatial learning/memory following repeated administration of α-PVP and the possible role of brain mitochondrial dysfunction in these cognitive impairments.

Stylopine: A potential natural metabolite to block vascular endothelial growth factor receptor 2 (VEGFR2) in osteosarcoma therapy.

Vascular endothelial growth factor (VEGF) signals cell survival, cell migration, osteogenesis, cell proliferation, angiogenesis, and vascular permeability by binding to VEGF receptor 2 (VEGFR-2). Osteosarcoma is the most common primary bone cancer, majorly affects young adults. Activation of VEGFR-2 signaling is a therapeutic target for osteosarcoma. The present study aimed to evaluate the potency of stylopine in regulation of the VEGFR-2 signaling pathway and its anti-tumour effect human MG-63 osteosarcoma cells. The in silico study on benzylisoquinoline alkaloids was carried out for analyzing and shortlisting of compounds using a virtual screening, Lipinski's rule, bioavailability graphical RADAR plot, pharmacokinetics, toxicity, and molecular docking studies. Among the benzylisoquinoline alkaloids, stylopine was selected and subjected to in-vitro studies against human MG-63 osteosarcoma cells. Various experiments such as MTT assay, EtBr/AO staining, mitochondrial membrane potential assessment, transwell migration assay, gene expression analysis by a quantitative real time polymerase chain reaction (qRT-PCR) method, SDS-PAGE followed by immunoblotting were performed to evaluate its anti-tumour effect as compared to standard axitinib. The MTT assay indicates that stylopine inhibits cell proliferation in MG-63 cells. Similarly, as confirmed by the EtBr/Ao staining method, the MMP assay indicates that stylopine induces mitochondrial membrane damage and apoptosis as compared to axitinib. Moreover, stylopine inhibits the VEGF-165 induced MG-63 cell migration by a trans-well migration assay. The immunoblotting and qRT-PCR analysis showed that stylopine inhibits the VEGF-165 induced VEGFR2 expression in MG-63 cells. It is concluded that stylopine has potential to regulate VEGFR2 and can inhibit osteosarcoma cells to offer a new drug candidate for the treatment of bone cancer in future.

A Concerted Redox- and Light-Activated Agent for Controlled Multimodal Therapy against Hypoxic Cancer Cells.

Hypoxia represents a remarkably exploitable target for cancer therapy, is encountered only in solid human tumors, and is highly associated with cancer resistance and recurrence. Here, a hypoxia-activated mitochondria-accumulated Ru(II) polypyridyl prodrug functionalized with conjugated azo (Az) and nitrogen mustard (NM) functionalities, RuAzNM, is reported. This prodrug has multimodal theranostic properties toward hypoxic cancer cells. Reduction of the azo group in hypoxic cell microenvironments gives rise to the generation of two primary amine products, a free aniline mustard, and the polypyridyl RuNH2 complex. Thus, the aniline mustard triggers generation ofreactive oxygen species (ROS) and mtDNA crosslinking. Meanwhile, the resultant biologically benign phosphorescent RuNH2 gives rise to a diagnostic signal and signals activation of the phototherapy. This multimodal therapeutic effect eventually elevates ROS levels, depletes reduced nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP), and induces mitochondrial membrane damage, mtDNA damage, and ultimately cell apoptosis. This unique strategy allows controlled multimodal theranostics to be realized in hypoxic cells and multicellular spheroids, making RuAzNM a highly selective and effective cancer-cell-selective theranostic agent (IC50 = 2.3µm for hypoxic HepG2 cancer cells vs 58.2µm for normoxic THL-3 normal cells). This is the first report of a metal-based compound developed as a multimodal theranostic agent for hypoxia.

Red light-activable biotinylated copper(II) complex-functionalized gold nanocomposite (Biotin-Cu@AuNP) towards targeted photodynamic therapy

We report the synthesis and characterization of red-light activable gold nanoparticle functionalized with biotinylated copper(II) complex of general molecular formula, [Cu(L3)(L6)]-AuNPs (Biotin-Cu@AuNP), where L3 = N-(3-((E)-3,5-di-tert-butyl-2-hydroxybenzylideneamino)-4-hydroxyphenyl)-5-((3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide, L6 = 5-(1,2-dithiolan-3-yl)-N-(1,10-phenanthrolin-5-yl)pentanamide, which was explored for their photophysical, theoretical and photo-cytotoxic potentials. The nanoconjugate exhibits differential uptake in biotin positive and biotin negative cancer cells as well as normal cells. The nanoconjugate also shows remarkable photodynamic activity against biotin positive A549 (IC50: 13 μg/mL in red light; >150 μg/mL in dark) and HaCaT (IC50: 23 μg/mL in red light; >150 μg/mL in dark) cells under red light (600–720 nm, 30 Jcm−2) irradiation, with significantly high photo-indices (PI>15). The nanoconjugate is less toxic to HEK293T (biotin negative) and HPL1D (normal) cells. Confocal microscopy confirms preferential mitochondrial and partly cytoplasmic localization of Biotin-Cu@AuNP in A549 cells. Several photo-physical and theoretical studies reveal the red light-assisted generation of singlet oxygen (1O2) (Ф (1O2) =0.68) as a reactive oxygen species (ROS) which results in remarkable oxidative stress and mitochondrial membrane damage, leading to caspase 3/7-dependent apoptosis of A549 cells. Overall, the nanocomposite (Biotin-Cu@AuNP) exhibiting red light-assisted targeted photodynamic activity has emerged as the ideal next generation PDT agents.