Avermectin B1a Shows Potential Anti-Proliferative and Anticancer Effects in HCT-116 Cells via Enhancing the Stability of Microtubules.

Avermectins are a group of macrocyclic lactones that are commonly used as pesticides to treat pests and parasitic worms. Some members of the avermectin family, such as ivermectin, have been found to exhibit anti-proliferative activity toward cancer cells. This study aimed to investigate the potential anti-cancer activities of avermectin B1a using the HCT-116 colon cancer cell line. The MTT assay was used to calculate the IC50 by incubating cells with increasing doses of avermectin B1a for 24, 48, and 72 h. Flow cytometry was used to evaluate apoptosis following the 24 h incubation of cells. The migration capacity of the HCT-116 cells in the absence or presence of avermectin B1a was also investigated. Finally, tubulin polymerization in the presence of avermectin B1a was evaluated. Avermectin B1a presented anti-proliferative activity with an IC50 value of 30 µM. Avermectin B1a was found to promote tubulin polymerization at 30 µM. In addition, avermectin B1a induced apoptosis in HCT-116 cells and substantially diminished their ability to migrate. Avermectin B1a exhibits significant anti-cancer activity and enhances tubulin polymerization, suggesting that it can be used as a promising microtubule-targeting agent for the development of future anticancer drugs.

Mechanistic insights into the inhibition of human placental glutathione S-transferase P1-1 by abscisic and gibberellic acids: An integrated experimental and computational study.

The interactions of the classic phytohormones gibberellic acid (gibberellin A3 , GA3 ) and abscisic acid (dormin, ABA), which antagonistically regulate several developmental processes and stress responses in higher plants, with human placental glutathione S-transferase P1-1 (hpGSTP1-1), an enzyme that plays a role in endo- or xenobiotic detoxification and regulation of cell survival and apoptosis, were investigated. The inhibitory potencies of ABA and GA3 against hpGSTP1, as well as the types of inhibition and the kinetic parameters, were determined by making use of both enzyme kinetic graphs and SPSS nonlinear regression models. The structural basis for the interaction between hpGSTP1-1 and phytohormones was predicted with the aid of molecular docking simulations. The IC50 values of ABA and GA3 were 5.3 and 5.0 mM, respectively. Both phytohormones inhibited hpGSTP1-1 in competitive manner with respect to the cosubstrates GSH and CDNB. When ABA was the inhibitor at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v , Vm , Km , and Ki values were statistically estimated to be 205 ± 16 µmol/min-mg protein, 1.32 ± 0.18 mM, 1.95 ± 0.25 mM and 175 ± 6 µmol/min-mg protein, 0.85 ± 0.06 mM, 1.85 ± 0.16 mM, respectively. On the other hand, the kinetic parameters Vm , Km , and Ki obtained with GA3 at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v were found to be 303 ± 14 µmol/min-mg protein, 1.77 ± 0.13 mM, 3.38 ± 0.26 mM and 249 ± 7 µmol/min-mg protein, 1.43 ± 0.07 mM, 2.89 ± 0.19 mM, respectively. Both phytohormones had the potential to engage in hydrogen-bonding and electrostatic interactions with the key residues that line the G- and H-sites of the enzyme's catalytic center. Inhibitory actions of ABA/GA3 on hpGSTP1-1 may guide medicinal chemists through the structure-based design of novel antineoplastic agents. It should be noted, however, that the same interactions may also render fetuses vulnerable to the potentially toxic effects of xenobiotics and noxious endobiotics.

Mesenchymal stem cell-derived exosomes as new tools for delivery of miRNAs in the treatment of cancer.

Although ongoing medical research is working to find a cure for a variety of cancers, it continues to be one of the major causes of death worldwide. Chemotherapy and immunotherapy, as well as surgical intervention and radiation therapy, are critical components of cancer treatment. Most anti-cancer drugs are given systemically and distribute not just to tumor tissues but also to normal tissues, where they may cause side effects. Furthermore, because anti-cancer drugs have a low delivery efficiency, some tumors do not respond to them. As a result, tumor-targeted drug delivery is critical for improving the safety and efficacy of anti-cancer treatment. Exosomes are microscopic extracellular vesicles that cells produce to communicate with one another. MicroRNA (miRNA), long non-coding RNA (lncRNA), small interfering RNA (siRNA), DNA, protein, and lipids are among the therapeutic cargos found in exosomes. Recently, several studies have focused on miRNAs as a potential therapeutic element for the treatment of cancer. Mesenchymal stem cells (MSC) have been known to have angiogenic, anti-apoptotic, anti-inflammatory and immunomodulatory effects. Exosomes derived from MSCs are gaining popularity as a non-cellular alternative to MSC-based therapy, as this method avoids unwanted lineage differentiation. Therefore more research have focused on transferring miRNAs to mesenchymal stem cells (MSC) and targeting miRNA-loaded exosomes to cancer cells. Here, we initially gave an overview of the characteristics and potentials of MSC as well as the use of MSC-derived exosomes in cancer therapy. Finally, we emphasized the utilization of MSC-derived exosomes for miRNA delivery in the treatment of cancer.

Evaluation of the penetration of intracoronal bleaching agents into the cervical region using different intraorifice barriers.

BACKGROUND: The present study aimed to make a comparison between the effects of 35% hydrogen peroxide gel (HP) and sodium perborate with distilled water (SP) bleaching agents on the sealing characteristics of glass ionomer cement (GIC), TheraBase, ProRoot MTA and Biodentine intraorifice barriers. METHODS: One hundred and twelve single-rooted mandibular human premolar teeth extracted from young patients (14-25 years) were chosen. Root cement and cementoenamel junction (CEJ) of teeth were examined under a stereomicroscope at 10 × magnification to ensure there was no cement defect or dentin gap in CEJ. After the endodontic access cavities were opened on the occlusal surfaces of the teeth, the working length was determined. Instrumentation of each root canal was performed with a ProTaper Gold rotary system in the determined working length and filled with gutta-percha + AH Plus with a single cone technique using. Root fillings were removed 3 mm short of the CEJ and sealed with one of the following intraorifice barrier materials (n = 30/group): 1. GIC; 2. TheraBase; 3. ProProot-MTA; 4. Biodentine. In each of the sub-groups, either HP or SP was used to perform intracoronal bleaching on days 1, 4, and 7. All outer surfaces of the specimens except the 3 mm cervical region were covered with nail polish and modeling wax layers. Specimens were immersed in a 5 ml Eppendorf tube that contained 2 mL of distilled water. The penetration of peroxide release was measured using the colorimetric ferric thiocyanate method. Statistical analysis of the data was performed with Three-way ANOVA and Tukey's test (P = 0.05). RESULTS: In the HP groups, GIC showed the greatest peroxide release when compared with other tested groups on day 1 (P < 0.05). Biodentine and ProRoot MTA displayed a significantly lower peroxide leakage when compared to GIC and TheraBase on days 1 and 4 (P < 0.05). While GIC and TheraBase were used, HP observed higher peroxide penetration when compared with SP on days 1 and 4 (P < 0.05). CONCLUSIONS: Peroxide diffusion was significantly influenced by the kind of intracoronal bleaching agents and intraorifice barrier materials used.

Enzalutamide Overcomes Dihydrotestosterone-Induced Chemoresistance in Triple- Negative Breast Cancer Cells via Apoptosis.

BACKGROUND: Triple-negative breast cancer is challenging to treat due to its heterogeneity and lack of therapeutic targets. Hence, systemic chemotherapy is still the mainstay in TNBC treatment. Unfortunately, patients commonly develop chemoresistance. Androgen signalling through its receptor is an essential player in breast cancer, where it has been shown to confer chemoresistance to TNBC cells. OBJECTIVE: The objective of the study was to elucidate the mechanistic effects of enzalutamide in the chemoresponse of TNBC cells to doxorubicin through the apoptosis pathway. METHODS: MDA-MB-231 and MDA-MB-453 cells were used as model systems of TNBC. Cell viability and apoptosis were investigated upon treatment of cells with doxorubicin in the presence of dihydrotestosterone (DHT) and/or enzalutamide. Caspase 3/7 activity and TUNEL assays were performed to assess the induction of apoptosis. The expression of apoptosis-regulatory genes was assayed by qPCR for the detection of expression changes. RESULTS: Enzalutamide decreased the viability of MDA-MB-231 and MDA-MB- 453 cells and reduced DHT-induced chemoresistance of both cell lines. It also increased the chemosensitivity towards doxorubicin in MDA-MB-231 cells. Increasing DNA degradation and caspase 3/7 activity were concomitant with these outcomes. Moreover, enzalutamide downregulated the expression of the anti-apoptosis genes, mcl1 and bcl2, in MDA-MB-231 cells, while increasing the expression of the pro-apoptotic gene bid. On the other hand, DHT upregulated the expression of the anti-apoptosis genes, mcl1 and bcl2, in both cell lines. CONCLUSION: DHT increased the expression of the anti-apoptosis genes mcl1 and bcl2 in the TNBC cells, presumably leading to cell survival via the prevention of doxorubicin-induced apoptosis. On the other hand, enzalutamide may sensitize the cells to doxorubicin through downregulation of the bid/bcl2/mcl1 axis that normally activates the executive caspases, caspase 3/7. The activities of the latter enzymes were apparent in DNA degradation at the late stages of apoptosis.

The Relevance of Glutathione Reductase Inhibition by Fluoxetine to Human Health and Disease: Insights Derived from a Combined Kinetic and Docking Study.

Glutathione reductase (GR) is a homodimeric enzyme playing an important role in the regeneration of the central antioxidant molecule reduced glutathione (GSH) from oxidized glutathione (GSSG) at the expense of a molecule of NADPH. GSH scavenges and eliminates superoxide and hydroxyl radicals non-enzymatically or serves as an electron donor for several enzymes. Fluoxetine (FLU), a selective serotonin reuptake inhibitor, is widely prescribed in the treatment of major depressive disorder. Here, using enzyme kinetic studies and molecular docking simulations, we aimed at disclosing the mechanistic and structural aspects of the interaction between GR and FLU. Affecting enzyme activity in a dose-dependent manner, FLU was shown to be a moderately potent (IC50 = 0.88 mM) inhibitor of GR. When the variable substrate was GSSG, the type of inhibition was linear mixed-type competitive (Ki = 279 ± 32 µM; α = 5.48 ± 1.29). When the variable substrate was NADPH, however, the type of inhibition was non-competitive (Ki = 879 ± 82 µM). The observed difference in inhibition types was attributed to the binding of FLU in the large intermonomer cavity of GR, where it hampered catalysis and interfered with substrate binding. Overall, although it is anticipated that long-term use of FLU leads to acquired GR deficiency, the inhibitory action of FLU on GR may be therapeutically exploited in anti-cancer research.

Assessment of the inhibitory activity of the pyrethroid pesticide deltamethrin against human placental glutathione transferase P1-1: A combined kinetic and docking study.

Deltamethrin (DEL), which is a synthetic pyrethroid insecticide, has been used successfully all over the world to treat mosquito nets for the control of malaria. Glutathione S-transferases (GSTs; EC 2.5.1.18) catalyze the conjugation of reduced glutathione (GSH) to a variety of xenobiotics and are normally recognized as detoxification enzymes. Here, we used a colorimetric assay based on the human placental GSTP1-1 (hpGSTP1-1)-catalyzed reaction between GSH and the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) as well as molecular docking to investigate the mechanistic and structural aspects of hpGSTP1-1 inhibition by DEL. We show that DEL is a potent, noncompetitive inhibitor of hpGSTP1-1 with an IC50 value of 6.1 µM and Ki values of 5.61 ±â€¯0.32 µM and 7.96 ±â€¯0.97 µM at fixed [CDNB]-varied [GSH] and fixed [GSH]-varied [CDNB], respectively. DEL appears to be accommodated well in an eccentric cavity located at the interface of the hpGSTP1-1 homodimer, presumably causing conformational changes to the enzyme's substrate-binding sites such that the enzyme is no longer able to transform GSH and CDNB effectively. Correspondingly, considerable maternal exposure to and subsequent accumulation of DEL may interfere with the proper development of the vulnerable fetus, possibly increasing the risk of developing congenital defects.

Mechanistic and structural insights into the in vitro inhibitory action of hypericin on glutathione reductase purified from baker's yeast.

This work aims at studying the interaction between glutathione reductase (GR) and hypericin. The type of inhibition was determined by measuring changes in GR activity at increasing concentrations of hypericin as well as at varying concentrations of glutathione disulfide (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH), and the binding pose of hypericin was predicted by molecular docking. Accordingly, hypericin emerges as an effective inhibitor of GR. When the variable substrate is GSSG, the type of inhibition is competitive. When the variable substrate is NADPH, however, the type of inhibition appears to be linear mixed-type competitive. Our computational analyses suggest that hypericin binds in the large intermonomer cavity of GR, and that it may interfere with the normal positioning/functioning of the redox-active disulfide center at the enzyme's active site. Overall, besides its contributory role in promoting oxidative stress via the formation of reactive oxygen species in photodynamic therapy, hypericin can also weaken cancer cells through inhibiting GR.

Purification of Glutathione S-Transferase pi from Erythrocytes and Evaluation of the Inhibitory Effect of Hypericin.

Hypericin is a photosensitizer compound used in the photodynamic therapy (PDT). PDT is an alternative cancer treatment strategy whose function is dependent on the photosensitizers accumulating selectively in tumor cells and following visible or infra-red light induced activation lead to the apoptosis/necrosis of the tumor cells via the formation of reactive oxygen species. Thus, the cellular redox balance is essential for the efficacy of PDT. Among the protective enzyme systems glutathione S-transferases (GST, E.C.2.5.1.18) function in detoxification, protection against oxidative stress and intracellular transport of molecules. It is known that isoenzymes of GST and especially GST-pi is increased in cancer cells and it plays very important functions in the development of resistance to anticancer drugs. Since photosensitizers are used intravenously, it is important to elucidate the effects of photosensitizers on the erythrocyte enzymes. The aim of the present study was to investigate the impact of hypericin on human erythrocyte GST-pi (heGST-pi). Purification yield of 71% and purification fold of 2550 were achieved by using conventional chromatographic methods. The specific activity of the enzyme is found as 51 U/mg protein. Hypericin inhibited heGST-pi in a dose dependent manner and inhibition was biphasic. Noncompetitive type of inhibition was observed with both substrates, GSH and CDNB. The inhibitory constant (K i ) values obtained from Lineweaver-Burk, Dixon, secondary plots; slope and y-intercept versus 1/S (substrate) and from non-linear regression analysis were in good correlation: K i (GSH) was calculated as 0.19 ± 0.01 µM and K i (CDNB) as 0.26 ± 0.03 µM.

Amitriptyline may have a supportive role in cancer treatment by inhibiting glutathione S-transferase pi (GST-π) and alpha (GST-α).

A tricyclic anti-depressant, amitriptyline, is a highly prescribed drug for cancer patients for mood elevation but there are limited studies about the interaction of amitriptyline with glutathione S-transferases pi (GST-π) and glutathione S-transferases alpha (GST-α). GST isozymes have been implicated in chemotherapeutic drug resistance. We demonstrated that the concentration dependent inhibition of GST-π and GST-α by amitriptyline followed inverse hyperbolic inhibition curves with IC(50) values of 5.54 and 8.32 mM, respectively. When the varied substrate was GSH, amitriptyline inhibited both isozymes competitively and similar K(i) values were found for GST-π (K(i) = 1.61 ± 0.17 mM) and GST-α (K(i) = 1.45 ± 0.20 mM). On the other hand, when the varied substrate was CDNB, the inhibition types were non-competitive for GST-π (K(i) = 1.98 ± 0.31 mM) and competitive for GST-α (K(i) = 1.57 ± 0.16 mM). Amitriptyline, in addition to its antidepressant effect, might also have a minor supportive role on the effectiveness of the anticancer drugs by decreasing their elimination through inhibiting GST-π and GST-α.

Evaluation of the in vitro inhibitory impact of hypericin on placental glutathione S-transferase pi.

St John's Wort (SJW) extracts are herbal products which are available without prescription in most countries and widely used in the treatment of mild to moderate depression. Since it is a herbal product and available without prescription, use of SJW is common among pregnant and/or lactating woman. The principal of the study was to clarify the effects of hypericin, one of the components of SJW, on glutathione S-transferase-pi (GST-pi) purified from human placenta. The K (m) values of GST-pi were 0.21 ± 0.03 mM for glutathione (GSH) and 2.29 ± 0.54 mM for 1-chloro-2,4-dinitrobenzene (CDNB). At fixed [GSH], the V (m) value calculated was about 3 times higher than the conditions in which [CDNB] was fixed; 201 ± 30 U/mg protein versus 74 ± 3 U/mg protein. At constant substrate concentrations (1 mM), an average IC (50) value of 0.70 ± 0.02 µM was obtained. Hypericin inhibited GST-pi competitively with respect to both substrates. When GSH was the varied substrate a K (i) value of 0.31 ± 0.05 µM was found; when CDNB was the varied substrate, a K (i) value of 0.85 ± 0.02 µM was obtained. On the basis of these data considering transplacental transfer of hypericin and immature hepatic clearance of the baby, using this herbal product may cause abnormalites due to the inhibition of one of the most important placental detoxification enzymes, GST-pi.

The inhibition characteristics of human placental glutathione S-transferase-π by tricyclic antidepressants: amitriptyline and clomipramine.

Tricyclic antidepressants (TCAs) are the non-selective amine re-uptake inhibitors, well absorbed from small intestine, cross the blood-brain barrier, distributed in the brain, and are bound to glutathione S-transferase-π (GST-π). TCAs can pass through placenta, accumulate in utero baby, and cause congenital malformations. Thus, the study of the interaction of GST-π with antidepressants is crucial. In this study, the interaction of GST-π with amitriptyline and clomipramine was investigated. The K (m) values for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) were found to be 0.16 ± 0.04 and 3.60 ± 1.67 mM, respectively. The V (m) values were varying according to the fixed substrate; [CDNB] fixed, 53 ± 3 and [GSH] fixed 182 ± 63 U/mg protein. At variable [GSH] and variable [CDNB], the k (cat) values of 7.0 × 10(6) and 1.42 × 10(7) s(-1) and the k (cat)/K (m) values of 4.38 × 10(10) and 3.94 × 10(9 )M(-1 )s(-1) were obtained, respectively. At fixed [CDNB] and variable [GSH], amitriptyline (K (s) = 0.16 ± 0.03 mM; α = 2.08; and K (i) = 1.75 ± 0.37 mM) and clomipramine (K (s) = 0.24 ± 0.05 mM; α = 1.57; and K (i) = 3.90 ± 2.26 mM) showed linear mixed-type inhibition whereas when the varied substrate is CDNB, amitriptyline (K (i) = 4.90 ± 0.68 mM) and clomipramine (K (i) = 3.37 ± 0.39 mM) inhibition were noncompetitive. The inhibition of GST-π by TCAs means the destruction of its protective role against toxic electrophiles. The effect of antidepressants on fetus will be much severe, thus, the antidepressant therapy of pregnant women should be done with caution.

Inhibition characteristics of hypericin on rat small intestine glutathione-S-transferases.

Glutathione-S-transferases constitute a family of enzymes involving in the detoxification of xenobiotics, signalling cascades and serving as ligandins or/and catalyzing the conjugation of various chemicals and drugs. The widely expressed cytosolic GST-pi is a marker protein in various cancers and its increased concentration is linked to drug resistance. GST-pi is autoregulated by S-glutathionylation and it catalyzes the S-glutathionylation of other proteins in response to oxidative or nitrosative stress. S-glutathionylation of GST-pi results in multimer formation and the breakage of ligand binding interactions with c-Jun NH(2)-terminal kinase (JNK). Another widely expressed GST enzyme, GST-alpha is assumed as a marker in hepatocellular damage, is implicated in cancer, asthma, cardiovascular disease and response to chemotherapy. Although, it was shown that hypericin binds and inhibits GST-alpha and GST-pi, the inhibition characteristics have not been investigated in detail. The aim of this study was to investigate the effects of hypericin on major GSTs; GST-alpha and GST-pi purified from rat small intestine. When GSH used as varied substrate the inhibition pattern with hypericin was uncompetitive for GST-alpha (K(i)=0.16 + or - 0.02 microM) and noncompetitive for GST-pi (K(i) = 2.46 + or - 0.43 microM). While using CDNB (1-chloro-2,4-dinitrobenzene) as the varied substrate, the inhibition patterns were noncompetitive for GST-alpha and competitive for GST-pi; K(i) values for GST-alpha and GST-pi were 1.91 + or - 0.21 and 0.55 + or - 0.07 microM, respectively. Since hypericin accumulated in cancer cells and important in photodynamic therapy (PDT), inhibition of GST-alpha and GST-pi by hypericin might increase the effectivity of the treatment. Considering that GST-pi is responsible for the drug resistance its inhibition might increase the benefit obtained from chemotherapy.

Insulin receptor substrate-1 regulates the transformed phenotype of BT-20 human mammary cancer cells.

Although originating from a human breast cancer, BT-20 cells do not form colonies in soft agar. BT-20 cells do not express insulin receptor substrate-1 (IRS-1), which is known to promote both normal and abnormal growth and to inhibit differentiation. Stable expression of IRS-1 confers to BT-20 cells the ability to form colonies in soft agar. BT-20 cells form tumors in xenografts in mice, but the size of tumors is twice as large when the cells express IRS-1. The increased transformed phenotype is characterized by occupancy of the rDNA and cyclin D1 promoters by IRS-1 and the activation of the cyclin D1, c-myc, and rDNA promoters. In addition, the retinoblastoma protein, which is detectable in the rDNA promoter of quiescent BT-20/IRS-1 cells, is replaced by IRS-1 after insulin-like growth factor-I stimulation. Our results indicate that in BT-20 human mammary cancer cells, expression of IRS-1 activates promoters involved in cell growth and cell proliferation, resulting in a more transformed phenotype. Targeting of IRS-1 could be effective in inhibiting the proliferation of mammary cancer cells.