In Vitro Antibacterial and Phytochemical Screening of Hypericum perforatum Extract as Potential Antimicrobial Agents against Multi-Drug-Resistant (MDR) Strains of Clinical Origin.

Background: The perennial plant Hypericum perforatum is widely distributed around the world. It has been used for many years in conventional medicine to treat a variety of illnesses, including stress, mild to moderate depression, and minor injuries. This study examined the antimicrobial activity of the H. perforatum total extract and its fractions (n-hexane, ethyl acetate, chloroform, and aqueous) against multi-drug-resistant (MDR) isolates that were gathered from clinical samples, including methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Escherichia coli, and Klebsiella pneumonia. Materials and Methods: Aerial parts of H. perforatum were collected and extracted using various solvents and were tested versus different isolated bacterial species. The inhibition zone of tested extracts was detected using an agar diffusion assay, and MICs were measured. Phytochemical analysis of promising H. perforatum extract was done using LC-ESI-MS/MS. Ultrastructure examination for the most altered bacteria used transmission electron microscopy. Antioxidant assays were done using DPPH and ABTS scavenging capacity methods. Cytotoxicity was reported versus Vero cells. Results: Different extracts of H. perforatum showed promising antibacterial activity against the pathogens. While the subfractions of the total extract were observed to show lesser inhibition zones and higher MIC values than the total extract of H. perforatum against MDR strains, the total extract of H. perforatum demonstrated the most potent antimicrobial action with an inhibition zone range of 17.9-27.9 mm. MDR-K. pneumoniae was discovered to be the most susceptible strain, which is consistent with the antibacterial inhibitory action of H. perforatum whole extract. Additionally, after treatment at the minimum inhibitory concentration (MIC 3.9 µg/ml), the transmission electron microscope showed alterations in the ultrastructure of the K. pneumoniae cells. Methanol extract from H. perforatum has a CC50 value of 976.75 µg/ml. Conclusion: Future inhibitors that target MDR strains may be revealed by these findings. Additionally, the extracts that were put to the test demonstrated strong antioxidant effects as shown by DPPH or ABTS radical-scavenging assays.

S-20, a steroidal saponin from the berries of black nightshade, exerts anti-multidrug resistance activity in K562/ADR cells through autophagic cell death and ERK activation.

Multidrug resistance (MDR) is a major cause of chemotherapy failure. Adriamycin (ADR) has been widely used to treat cancer, however, as a substrate of the adenosine triphosphate binding cassette (ABC) transporter, it is easy to develop drug resistance during the treatment. Here, we demonstrated that steroidal saponin S-20 isolated from the berries of black nightshade has comparable cytotoxicity in ADR-sensitive and resistant K562 cell lines. Autophagy is generally considered to be a protective mechanism to mediate MDR during treatment. However, we found that S-20-induced cell death in K562/ADR is associated with autophagy. We further explored the underlying mechanisms and found that S-20 induces caspase-dependent apoptosis in ADR-sensitive and resistant K562 cell lines. Most importantly, S-20-induced autophagy activates the ERK pathway and then inhibits the expression of drug resistance protein, which is the main reason to overcome K562/ADR resistance, rather than apoptosis. Taken together, our findings emphasize that S-20 exerts anti-multidrug resistance activity in K562/ADR cells through autophagic cell death and ERK activation, which may be considered as an effective strategy.

A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2).

The ABCG2 transporter plays a pivotal role in multidrug resistance, however, no clinical trial using specific ABCG2 inhibitors have been successful. Although ABC transporters actively extrude a wide variety of substrates, photodynamic therapeutic agents with porphyrinic scaffolds are exclusively transported by ABCG2. In this work, we describe for the first time a porphyrin derivative (4B) inhibitor of ABCG2 and capable to overcome multidrug resistance in vitro. The inhibition was time-dependent and 4B was not itself transported by ABCG2. Independently of the substrate, the porphyrin 4B showed an IC50 value of 1.6 µM and a mixed type of inhibition. This compound inhibited the ATPase activity and increased the binding of the conformational-sensitive antibody 5D3. A thermostability assay confirmed allosteric protein changes triggered by the porphyrin. Long-timescale molecular dynamics simulations revealed a different behavior between the ABCG2 porphyrinic substrate pheophorbide a and the porphyrin 4B. Pheophorbide a was able to bind in three different protein sites but 4B showed one binding conformation with a strong ionic interaction with GLU446. The inhibition was selective toward ABCG2, since no inhibition was observed for P-glycoprotein and MRP1. Finally, this compound successfully chemosensitized cells that overexpress ABCG2. These findings reinforce that substrates may be a privileged source of chemical scaffolds for identification of new inhibitors of multidrug resistance-linked ABC transporters.

Magnolol and Honokiol Inhibited the Function and Expression of BCRP with Mechanism Exploration.

Breast cancer resistance protein (BCRP), one of the ATP-binding cassette (ABC) transporters, was associated with the multidrug resistance (MDR) of chemotherapy. Magnolol (MN) and honokiol (HK) are major bioactive polyphenols of Magnolia officinalis. This study investigated the effects of MN and HK on the function and expression of BCRP for the purpose of developing BCRP inhibitor to overcome MDR. Cell lines including MDCKII-BCRP and MDCKII-WT were used for evaluating the function and expression of BCRP. The results showed that MN (100-12.5 µM) and HK (100-12.5 µM) significantly decreased the function of BCRP by 80~12% and 67~14%, respectively. In addition, MN and HK were verified as substrates of BCRP. Furthermore, MN and HK reduced the protein expression of BCRP, and inhibited the phosphorylation of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K). In conclusion, both MN and HK decreased the function and expression of BCRP via EGFR/PI3K signaling pathway. Therefore, both compounds were promising candidates for reversing the MDR of chemotherapy.

Photodynamic disinfection and its role in controlling infectious diseases.

Photodynamic therapy is witnessing a revival of its origins as a response to the rise of multi-drug resistant infections and the shortage of new classes of antibiotics. Photodynamic disinfection (PDDI) of microorganisms is making progresses in preclinical models and in clinical cases, and the perception of its role in the clinical armamentarium for the management of infectious diseases is changing. We review the positioning of PDDI from the perspective of its ability to respond to clinical needs. Emphasis is placed on the pipeline of photosensitizers that proved effective to inactivate biofilms, showed efficacy in animal models of infectious diseases or reached clinical trials. Novel opportunities resulting from the COVID-19 pandemic are briefly discussed. The molecular features of promising photosensitizers are emphasized and contrasted with those of photosensitizers used in the treatment of solid tumors. The development of photosensitizers has been accompanied by the fabrication of a variety of affordable and customizable light sources. We critically discuss the combination between photosensitizer and light source properties that may leverage PDDI and expand its applications to wider markets. The success of PDDI in the management of infectious diseases will ultimately depend on the efficacy of photosensitizers, affordability of the light sources, simplicity of the procedures, and availability of fast and efficient treatments.

A multifunctional nano-delivery system enhances the chemo-co-phototherapy of tumor multidrug resistance via mitochondrial-targeting and inhibiting P-glycoprotein-mediated efflux.

Despite the excellent progress of chemotherapy and phototherapy in tumor treatment, their effectiveness on multidrug-resistant (MDR) tumors is still unsatisfactory. One of the main obstacles is drug efflux caused by P-glycoprotein in MDR cells. Herein, we developed a nano-delivery system that combines a P-glycoprotein inhibitor with chemotherapy and phototherapy to overcome MDR. Briefly, the system is prepared by the self-assembly of a ROS-triggered doxorubicin prodrug (PTD) and mitochondrial-targeted D-α-tocopherol polyethyleneglycol succinate (TPP-TPGS), in which a photoactive drug, IR780, is encapsulated (PTD/TT/IR780). PTD/TT/IR780 can target the release of TPP-TPGS, doxorubicin and IR780 at the mitochondrial site of MDR cells through ROS trigger. D-α-Tocopherol polyethyleneglycol succinate (TPGS) is a P-glycoprotein inhibitor, which will reduce the efflux of doxorubicin and IR780 from MDR cells. Under irradiation of an 808 nm near-infrared laser, IR780 generates heat and ROS, causing mitochondrial damage and prompting MDR cell apoptosis. At the same time, ROS can reduce the ATP content, which inhibits the P-glycoprotein function. In addition, an increase in the ROS generates positive feedback, allowing more nanoparticles to be cleaved and further promoting payload release in MDR cells, thereby enhancing the synergistic efficacy of chemotherapy and phototherapy. The in vitro cellular assay showed that PTD/TT/IR780 significantly inhibited MDR cell proliferation at a very low drug concentration (IC50 = 0.27 µg mL-1 doxorubicin-equivalent concentration). In vivo animal experiments based on BALB/c nude mice bearing MCF-7/ADR tumors confirmed a superior antitumor efficacy and an excellent biosafety profile. These findings demonstrate that this multifunctional nanoplatform provides a new approach for the treatment of MDR tumors.

Elucidating the anticancer activities of guanidinium-functionalized amphiphilic random copolymers by varying the structure and composition in the hydrophobic monomer.

Rationale: Use of traditional anticancer chemotherapeutics has been hindered by the multifactorial nature of multi-drug resistance (MDR) development and metastasis. Recently, cationic polycarbonates were reported as novel unconventional anticancer agents that mitigated MDR and inhibited metastasis. The aim of this study is to explore structure-anticancer activity relationship. Specifically, a series of cationic guanidinium-based random copolymers of varying hydrophobicity was synthesized with a narrow polydispersity (Ð = 1.12-1.27) via organocatalytic ring-opening polymerization (OROP) of functional cyclic carbonate monomers, and evaluated for anticancer activity, killing kinetics, degradability and functional mechanism. Methods: Linear, branched and aromatic hydrophobic side chain units, such as ethyl, benzyl, butyl, isobutyl and hexyl moieties were explored as comonomer units for modulating anticancer activity. As hydrophobicity/hydrophilicity balance of the polymers determines their anticancer efficacy, the feed ratio between the two monomers was varied to tune their hydrophobicity. Results: Notably, incorporating the hexyl moiety greatly enhanced anticancer efficiency and killing kinetics on cancer cells. Degradation studies showed that the polymers degraded completely within 4-6 days. Flow cytometry and lactate dehydrogenase (LDH) release analyses demonstrated that anticancer mechanism of the copolymers containing a hydrophobic co-monomer was concentration dependent, apoptosis at IC50, and both apoptosis and necrosis at 2 × IC50. In contrast, the homopolymer without a hydrophobic comonomer killed cancer cells predominantly via apoptotic mechanism. Conclusion: The hydrophobicity of the polymers played an important role in anticancer efficacy, killing kinetics and anticancer mechanism. This study provides valuable insights into designing novel anticancer agents utilizing polymers.

Is Multidrug Resistance in Acute Otitis Media with Streptococcus pneumoniae Associated with a More Severe Disease?

OBJECTIVES: Streptococcus pneumoniae is the leading bacterial etiologic agent in acute otitis media (AOM), and it produces a more severe inflammatory response than other otopathogens. Additionally, the presence of multidrug-resistant (MDR) S. pneumoniae is an important issue in the management of AOM. The present pilot study aimed to ascertain whether MDR S. pneumoniae is associated with a higher inflammatory response and/or a more severe disease. METHODS: This was a prospective, single-center study on nonpneumococcal conjugate vaccine-immunized pediatric patients with severe AOM. Demographic and clinical characteristics were recorded. Middle ear fluid was obtained and cultured for each patient; antibiotic-resistance profiling was tested for S. pneumoniae isolates. The C-reactive protein (CRP) level and complete blood count were determined. Patients with positive middle ear fluid culture for S. pneumoniae were divided into 2 groups according to antibiotic resistance profile: MDR and non-MDR. RESULTS: MDR S. pneumoniae was identified in 15 (35.7%) of the 42 eligible patients. Children in this group had significantly higher CRP levels (72.23 ± 62.92 vs. 14.96 ± 15.57 mg/L, p < 0.001), higher absolute neutrophil count (8.46 ± 3.97 vs. 5.22 ± 4.5 × 103/mm3, p = 0.004), higher percentage of neutrophils (52.85 ± 13.49% vs. 38.34 ± 16.16%, p = 0.004), and were more prone to develop acute mastoiditis (p = 0.01). Receiver operating characteristic analysis identified CRP as the best biomarker to discriminate between the 2 groups of patients (AUC = 0.891). CONCLUSION: MDR S. pneumoniae was associated with a more severe inflammatory response and a higher incidence of mastoiditis.

Euphorbia Diterpenes: An Update of Isolation, Structure, Pharmacological Activities and Structure-Activity Relationship.

Euphorbia species have a rich history of ethnomedicinal use and ethnopharmacological applications in drug discovery. This is due to the presence of a wide range of diterpenes exhibiting great structural diversity and pharmacological activities. As a result, Euphorbia diterpenes have remained the focus of drug discovery investigations from natural products. The current review documents over 350 diterpenes, isolated from Euphorbia species, their structures, classification, biosynthetic pathways, and their structure-activity relationships for the period covering 2013-2020. Among the isolated diterpenes, over 20 skeletal structures were identified. Lathyrane, jatrophane, ingenane, ingenol, and ingol were identified as the major diterpenes in most Euphorbia species. Most of the isolated diterpenes were evaluated for their cytotoxicity activities, multidrug resistance abilities, and inhibitory activities in vitro, and reported good activities with significant half-inhibitory concentration (IC50) values ranging from 10-50 µM. The lathyranes, isopimaranes, and jatrophanes diterpenes were further found to show potent inhibition of P-glycoprotein, which is known to confer drug resistance abilities in cells leading to decreased cytotoxic effects. Structure-activity relationship (SAR) studies revealed the significance of a free hydroxyl group at position C-3 in enhancing the anticancer and anti-inflammatory activities and the negative effect it has in position C-2. Esterification of this functionality, in selected diterpenes, was found to enhance these activities. Thus, Euphorbia diterpenes offer a valuable source of lead compounds that could be investigated further as potential candidates for drug discovery.

Fatty Acids-Enriched Fractions of Hermetia illucens (Black Soldier Fly) Larvae Fat Can Combat MDR Pathogenic Fish Bacteria Aeromonas spp.

Aeromonas spp. cause many diseases in aquaculture habitats. Hermetia illucens (Hi) larvae were used as feed-in aquacultures and in eradicating pathogenic fish bacteria. In the present study, we applied consecutive extractions of the same biomass of BSFL fat using the acidic water-methanol solution. The major constituents of the sequential extracts (SEs) were free fatty acids (FFAs), and fatty acids derivatives as identified by gas chromatography spectrometry (GC-MS). Our improved procedure enabled gradual enrichment in the unsaturated fatty acids (USFAs) content in our SEs. The present study aimed to compare the composition and antimicrobial properties of SEs. Among actual fish pathogens, A. hydrophila and A. salmonicida demonstrated multiple drug resistance (MDR) against different recommended standard antibiotics: A. salmonicida was resistant to six, while A. hydrophila was resistant to four antibiotics from ten used in the present study. For the first time, we demonstrated the high dose-dependent antibacterial activity of each SE against Aeromonas spp., especially MDR A. salmonicida. The bacteriostatic and bactericidal (MIC/MBC) activity of SEs was significantly enhanced through the sequential extractions. The third sequential extract (AWME3) possessed the highest activity against Aeromonas spp.: inhibition zone diameters were in the range (21.47 ± 0.14-20.83 ± 0.22 mm) at a concentration of 40 mg/mL, MIC values ranged between 0.09 and 0.38 mg/mL for A. hydrophila and A. salmonicida, respectively. AWME3 MBC values recorded 0.19 and 0.38 mg/mL, while MIC50 values were 0.065 ± 0.004 and 0.22 ± 0.005 mg/mL against A. hydrophila and A. salmonicida, respectively. Thus, the larvae fat from Hermitia illucens may serve as an excellent reservoir of bioactive molecules with good capacity to eradicate the multidrug-resistant bacteria, having promising potential for practical application in the aquaculture field.

Curcumin at Low Doses Potentiates and at High Doses Inhibits ABT-737-Induced Platelet Apoptosis.

Curcumin is a natural bioactive component derived from the turmeric plant Curcuma longa, which exhibits a range of beneficial activities on human cells. Previously, an inhibitory effect of curcumin on platelets was demonstrated. However, it is unknown whether this inhibitory effect is due to platelet apoptosis or procoagulant platelet formation. In this study, curcumin did not activate caspase 3-dependent apoptosis of human platelets, but rather induced the formation of procoagulant platelets. Interestingly, curcumin at low concentration (5 µM) potentiated, and at high concentration (50 µM) inhibited ABT-737-induced platelet apoptosis, which was accompanied by inhibition of ABT-737-mediated thrombin generation. Platelet viability was not affected by curcumin at low concentration and was reduced by 17% at high concentration. Furthermore, curcumin-induced autophagy in human platelets via increased translocation of LC3I to LC3II, which was associated with activation of adenosine monophosphate (AMP) kinase and inhibition of protein kinase B activity. Because curcumin inhibits P-glycoprotein (P-gp) in cancer cells and contributes to overcoming multidrug resistance, we showed that curcumin similarly inhibited platelet P-gp activity. Our results revealed that the platelet inhibitory effect of curcumin is mediated by complex processes, including procoagulant platelet formation. Thus, curcumin may protect against or enhance caspase-dependent apoptosis in platelets under certain conditions.

Essential Oils, Pituranthos chloranthus and Teucrium ramosissimum, Chemosensitize Resistant Human Uterine Sarcoma MES-SA/Dx5 Cells to Doxorubicin by Inducing Apoptosis and Targeting P-Glycoprotein.

The multidrug resistance phenotype is a global phenomenon and causes chemotherapy failure in various cancers, such as in uterine sarcomas that have a high mortality rate. To overcome this phenotype, there is growing research interest in developing new treatment strategies. In this study, we highlight the potential of two essential oils from the Apiaceae family, Pituranthos chloranthus (PC) and Teucrium ramosissimum Desf. (TR), to act as chemopreventive and chemosensitizing agents against two uterine sarcoma cell lines, MES-SA and P-gp-overexpressing MES-SA/Dx5 cells. We found that PC and TR were able to inhibit the cell viability of sensitive MES-SA and resistant MES-SA/Dx5 cells by a slight modulation of the cell cycle and its regulators, but also through a significant induction of apoptosis. The molecular mechanism involved both caspase pathways associated with an overproduction of reactive oxygen species (ROS) and mitochondrial membrane depolarization. Very interestingly, the combination of doxorubicin with PC or TR induced a synergism to increase cell death in resistant MES-SA/Dx5 cells and, subsequently, had the benefit of decreasing the resistance index to doxorubicin. These synergistic effects were reinforced by a decrease in P-gp expression and its P-gp adenosine triphosphatase (ATPase) activity, which subsequently led to intracellular doxorubicin accumulation in resistant sarcoma cells.

Nanomedicines for combating multidrug resistance of cancer.

Chemotherapy typically involves the use of specific chemodrugs to inhibit the proliferation of cancer cells, but the frequent emergence of a variety of multidrug-resistant cancer cells poses a tremendous threat to our combat against cancer. The fundamental causes of multidrug resistance (MDR) have been studied for decades, and can be generally classified into two types: one is associated with the activation of diverse drug efflux pumps, which are responsible for translocating intracellular drug molecules out of the cells; the other is linked with some non-efflux pump-related mechanisms, such as antiapoptotic defense, enhanced DNA repair ability, and powerful antioxidant systems. To overcome MDR, intense efforts have been made to develop synergistic therapeutic strategies by introducing MDR inhibitors or combining chemotherapy with other therapeutic modalities, such as phototherapy, gene therapy, and gas therapy, in the hope that the drug-resistant cells can be sensitized toward chemotherapeutics. In particular, nanotechnology-based drug delivery platforms have shown the potential to integrate multiple therapeutic agents into one system. In this review, the focus was on the recent development of nanostrategies aiming to enhance the efficiency of chemotherapy and overcome the MDR of cancer in a synergistic manner. Different combinatorial strategies are introduced in detail and the advantages as well as underlying mechanisms of why these strategies can counteract MDR are discussed. This review is expected to shed new light on the design of advanced nanomedicines from the angle of materials and to deepen our understanding of MDR for the development of more effective anticancer strategies. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Antimicrobial activities evaluation and phytochemical screening of some selected medicinal plants: A possible alternative in the treatment of multidrug-resistant microbes.

BACKGROUND: Four out of five individuals rely on traditional medicine for their primary healthcare needs. Medicinal plants are endowed with diverse bioactive compounds to treat multidrug-resistant (MDR) microbes. So far, a less thorough examination has been made in this regard. This study aimed to evaluate antimicrobial activity and phytochemical screening of selected medicinal plants against MDR microbes. METHODS: In vitro experimental study was carried out to evaluate antimicrobial effects and phytochemical screening of Rumex abyssinicus, Cucumis pustulatus, Discopodium penninervium, Lippia adoensis, Euphorbia depauperata, Cirsium englerianum, and Polysphaeria aethiopica against MDR bacteria and fungi. Aqueous and 80% methanolic extraction methods were employed for extraction. The susceptibility test, minimum inhibitory concentration, and minimum bactericidal or fungicidal concentration were measured using disc diffusion or broth micro-dilution as per the CLSI protocols. RESULT: The 80% methanolic extraction method was a preferred method to aqueous. The phytochemical constituents identified were alkaloids, flavonoids, saponins, phenolic, tannins, terpenoidss, and cardiac glycosides. The hydroalcoholic extract demonstrated an appreciable antimicrobial role against MDR microbes with an MIC value of 1.0-128.0µg/ml and 11-29mm inhibition zone (IZ) in diameter. Extracts obtained from C. englerianum and E. depauperata showed a significant IZ ranged of 26-29mm on MRSA and Streptococcus pyogenes. MDR E. coli and K. pneumoniae showed 12-25mm and 23-28mm IZ in diameter, respectively. T. mentagraphytes was susceptible to all tested extracts. Moreover, S. pyogenes and K. pneumoniae were found the most susceptible bacteria to C. englerianum. Cirsium englerianum, L. adoensis, D. penninervium, and R. abyssinicus demonstrated remarkable antifungal effect against C. albicans and T. mentagrophytes, while R. abyssinicus showed the leading antifungal effect with 32 to 64µg/ml MIC values. CONCLUSION: The plant extracts have shown appreciable antimicrobial activities comparable to the currently prescribed modern drugs tested. Accordingly, further studies on clinical efficacy trial, safety, toxicity and affordability analyses have to be instigated promptly, so as to head to the final step to synthesize precursor molecules for new effective antimicrobials.

A novel flavonoid from Fissistigma cupreonitens, 5­hydroxy­7,8­dimethoxyflavanone, competitively inhibited the efflux function of human P-glycoprotein and reversed cancer multi-drug resistance.

BACKGROUND: P-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems. PURPOSE AND STUDY DESIGN: The aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well. METHODS: Calcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses. RESULTS: 5­hydroxy­7,8­dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 µg/ml. 5­hydroxy­7,8­dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 µg/ml). The docking results showed that 5­hydroxy­7,8­dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 µg/ml 5­hydroxy­7,8­dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively. CONCLUSION: The present study demonstrated that 5­hydroxy­7,8­dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion.

Evaluation of ethnopharmacologically selected Vitex negundo L. for In vitro antimalarial activity and secondary metabolite profiling.

ETHANOPHARMACOLOGICAL RELEVANCE: Limited drugs, rise in drug resistance against frontline anti-malarial drugs, non-availability of efficacious vaccines and high cost of drug development hinders malaria intervention programs. Search for safe, effective and affordable plant based anti-malarial agents, thus becomes crucial and vital in the current scenario. The Vitex negundo L. is medicinal plant possessing a variety of pharmaceutically important compounds. The plant is used traditionally worldwide for the treatment of malaria including India and Malaysia by the indigenous tribes. In vitro studies have reported the anti-malarial use of the plant in traditional medicinal systems. AIM OF THE STUDY: The aim of the current study is to evaluate the traditionally used medicinal plants for in vitro anti-malarial activity against human malaria parasite Plasmodium falciparum and profiling secondary metabolite using spectroscopic and chromatographic methods. Chemical profiling of active secondary metabolites in the extracts was undertaken using LC-MS. MATERIALS AND METHODS: Based on the ethno-botanical data V. negundo L. was selected for in vitro anti-malarial activity against P. falciparum chloroquine-sensitive (3D7) and multidrug resistant (K1) strains using SYBR Green-I based fluorescence assay. Cytotoxicity of extracts was evaluated in VERO cell line using the MTT assay. Haemolysis assay was performed using human red blood cells. Secondary metabolites profiling was undertaken using chromatographic and spectroscopic analysis. Liquid chromatography analysis was performed using a C18, 150 X 2.1, 2.6 µm column with gradient mobile phase Solvent A: 95% (H2O: ACN), Solvent B: Acetonitrile, Solvent C: Methanol, Solvent D: 5 mM NH4 in 95:5 (H2O: ACN) at a constant flow rate of 0.250 ml/min. The LC-MS spectra were acquired in both positive and negative ion modes with electrospray ionization (ESI) source. RESULTS: The anti-malarial active extract of V. negundo L. leaf exhibited potent anti-malarial activity with IC50 values of 7.21 µg/ml and 7.43 µg/ml against 3D7 and K1 strains, respectively with no evidence of significant cytotoxicity against mammalian cell line (VERO) and no toxicity as observed in haemolysis assay. The HPLC-LC-MS analysis of the extract led to identification of 73 compounds. We report for the first time the presence of Sabinene hydrate acetate, 5-Hydroxyoxindole, 2(3,4-dimethoxyphenyl)-6, 7-dimethoxychromen-4-one, Cyclotetracosa-1, 13-diene and 5, 7-Dimethoxyflavanone in the anti-malarial active extract of V. negundo L. leaf. Agnuside, Behenic acid and Globulol are some of the novel compounds with no reports of anti-malarial activity so far and require further evaluation in pure form for the development of potent anti-malarial compounds. CONCLUSIONS: The result report and scientifically validate the traditional use of V. negundo L. for the treatment of malaria providing new avenues for anti-malarial drug development. Several novel and unknown compounds were identified that need to be further characterized for anti-malarial potential.

Functional Doxorubicin-Loaded Omega-3 Unsaturated Fatty Acids Nanoparticles in Reversing Hepatocellular Carcinoma Multidrug Resistance.

BACKGROUND This study investigated a nanoparticle drug delivery system to reverse multidrug resistance (MDR) and assessed its anticancer efficacy in hepatocellular carcinoma (HCC). MATERIAL AND METHODS Docosahexaenoic acid (DHA) was used as the functional excipient and doxorubicin (DOX) as the chemotherapeutic drug to synthesize DOX nanoparticles (DOX-nano). The human HCC cell line HepG2 was used for experiments. HepG2/DOX, HepG2+DOX, HepG2+DOX-nano, HepG2/DOX+DOX, and HepG2/DOX+DOX-nano groups cells were treated with DOX or DOX-nano (5 µg/mL). Nude mice bearing a HepG2/DOX xenograft were divided into model, DOX, vector-nano, and DOX-nano groups and injected with saline, DOX reagent, vector-nano, and DOX-nano (2 mg/kg), respectively. Next, cytotoxicity, cellular uptake, cell apoptosis and migration, fluorescence imaging, TUNEL assay, and tumor inhibition effects were assessed in vitro and in vivo. Furthermore, expression of MDR-related proteins was also detected using western blotting. RESULTS Fluorescence imaging showed that the DOX uptake in the DOX-nano-treated group was the strongest in the HCC cells or tumors. Cell apoptosis was significantly increased in DOX-nano-treated HepG2/DOX cells and tumors, and cell migration was significantly inhibited in the DOX-nano-treated HepG2/DOX cells compared with the other groups. The tumor inhibitory rate in DOX-nano-injected tumors was also significantly higher than in other groups. The expression of breast cancer resistance protein, B-cell lymphoma 2, lung resistance protein, multidrug resistance protein, and protein kinase C alpha was significantly decreased in DOX-nano-treated HepG2/DOX cells and xenograft tumors. Significantly better antitumor and MDR-reversing effects were also observed in the HepG2+DOX group compared with the HepG2/DOX group. CONCLUSIONS This study revealed the potential efficacy of a DOX-nano drug delivery system for the treatment of HCC, using HepG2/DOX cells and nude mice bearing HepG2/DOX xenografts.

Stigmastane-type steroid saponins from the leaves of Vernonia amygdalina Del.

Vernoramyosides A-F (1-6), six new Δ7,9(11) stigmastane-type steroid saponins, along with four known analogues (7-10) were isolated from the leaves of Vernonia amygdalina Delile (Compositae). Their structures were determined by the combination of NMR, ECD and HR-ESI-MS data. These compounds all possessed highly oxidized side chain and a γ-lactam or α,ß-unsaturated five-membered lactone ring. All isolates were screened for their activities in reversing resistance in MCF/DOX cells.

Dihydromyricetin reverses MRP2-induced multidrug resistance by preventing NF-κB-Nrf2 signaling in colorectal cancer cell.

BACKGROUD: Dihydromyricetin (DMY), a natural flavonoid compound from the leaves of the Chinese medicinal herb Vitis heyneana, has been shown to have the potential to combat chemoresistance by inhibiting Nrf2/MRP2 signaling in colorectal cancer (CRC) cells. However, the precise underlying molecular mechanism and its therapeutic target are not well understood. PURPOSE: Our study aims to investigate the effects of DMY on multidrug resistance (MDR), and elucidate the underlying mechanisms. STUDY DESIGN: In vitro, HCT116/OXA and HCT8/VCR cells were employed as our MDR models. The cells were treated with DMY (50 µM) or MK-571 (50 µM) plus oxaliplatin (OXA) (10 µM) or vincristine (VCR) (10 µM) for 48 h. In vivo, we used BALB/c mice as a CRC xenograft mouse model. BALB/c mice were given DMY (100 mg/kg), OXA (5 mg/kg) and DMY (100 mg/kg) combined with OXA (5 mg/kg) via intraperitoneal route every 2 days per week for 4 weeks. METHODS: We used MTT and colony forming assays to detect DMY's ability to reverse MDR. Flow cytometric analysis was used to detect apoptosis. Immunocytochemistry was used to detect the localization of Nrf2 and NF-κB/p65. Western blot, qRT-PCR and reporter gene assays were employed to measure the protein and gene transcriptional levels (MRP2, Nrf2, NF-κB/p65). Moreover, chromatin immunoprecipitation (ChIP) assay was used to investigate the endogenous promoter occupancy of NF-κB/p65. Finally, immunohistochemistry and TUNEL staining were used to detect protein expression and apoptosis in vivo. RESULTS: DMY restored chemosensitivity (OXA and VCR) by inhibiting both MRP2 expression and its promoter activity in HCT116/OXA and HCT8/VCR cell lines. Furthermore, DMY could inhibit NF-κB/p65 expression, reducing NF-κB/p65 translocation to the nucleus to silence Nrf2 signaling, which is necessary for MRP2 expression. Overexpressing NF-κB/p65 expression reduced the reversal effect of DMY. In addition, NF-κB/p65 regulated Nrf2 expression by directly binding to its specific promoter region and activating its transcription. Finally, we proved that the combination of OXA and DMY has a synergistic tumor suppression effect in vivo. CONCLUSION: Our study provided a novel mechanism of DMY boosted chemosensitivity in human CRC. The downstream signals of DMY, NF-κB or Nrf2 could also be potential targets for the treatment of CRC.

NIR-II light triggered nitric oxide release nanoplatform combined chemo-photothermal therapy for overcoming multidrug resistant cancer.

The overexpression of P-glycoprotein (P-gp) in multidrug resistance (MDR) cancer cells increases the efflux of anticancer drugs thereby causing the failure of clinical chemotherapy. To address this obstacle, in this study, we rationally designed a near-infrared (NIR) light-responsive nitric oxide (NO) delivery nanoplatform for targeting the MDR tumors based on core-shell structured nanocomposites. The mesoporous silica shell provided abundant sites for modification of the NO donor, N-diazeniumdiolate, and tumor-targeting molecule, folic acid (FA), and enabled high encapsulation capacity for doxorubicin (DOX) loading. Under NIR light irradiation, the generation of NO gas can efficiently augment chemotherapeutic effects via the inhibition of P-gp expression. Simultaneously, the photothermal conversion agents of the Cu2-xSe core produce a large amount of heat for photothermal therapy (PTT). Finally, this combinational gas/chemo/PTT not only displays a superior and synergistic effect for overcoming MDR cancer, but also provides an efficient strategy to construct a multifunctional nano-drug delivery system with diversified therapeutic modalities.