Gossypol Acetic Acid Research Articles

Silica Gel Chromatographic Methods for Identification, Isolation and Purification of Gossypol Acetic Acid.

Several cottonseed varieties are cultivated in different countries. Each variety produces a different amount of gossypol as a natural toxic compound. The rising interest in cottonseed products (oil and feed) increases the demand for establishing simple methods for gossypol detection. Silica gel-based methods are ideal for its isolation, purification, and characterization. Silica gel-based methods are variants and can be used as simple methods for tracking plants' compounds. In this study, gossypol was isolated, characterized, and purified as gossypol acetic acid in the form of yellow crystals. Methods used for its characterization were TLC, preparative TLC, silica gel column, UV/IR spectrophotometer, and HPLC (robust spherical silica gel). A comparative study between its amount in both the Egyptian and Chinese varieties was performed. Under the experimental conditions, the Egyptian's cottonseed contains 8.705 gm/kg, while the Chinese's cottonseed contains 5.395 gm/kg. The TLC used in this study proved to be fast, accurate, and inexpensive. It can be used for gossypol acetic acid evaluation and quantification. Additionally, using TLC as a pre-purification step will give a pre-judgment for the sample's purity and quality. This step will protect the expensive HPLC silica gel-based column from any unexpected impurities. During each step, the silica gel itself could be simply removed by paper filtration. Collectively, the different silica gel-based methods as well as the other used methods are recommended for better Gossypol acetic acid isolation, purification, and characterization, as well as for maintaining HPLC columns.

Gossypol acetic acid regulates leukemia stem cells by degrading LRPPRC via inhibiting IL-6/JAK1/STAT3 signaling or resulting mitochondrial dysfunction.

Leukemia stem cells (LSCs) are found to be one of the main factors contributing to poor therapeutic effects in acute myeloid leukemia (AML), as they are protected by the bone marrow microenvironment (BMM) against conventional therapies. Gossypol acetic acid (GAA), which is extracted from the seeds of cotton plants, exerts anti-tumor roles in several types of cancer and has been reported to induce apoptosis of LSCs by inhibiting Bcl2. To investigate the exact roles of GAA in regulating LSCs under different microenvironments and the exact mechanism. In this study, LSCs were magnetically sorted from AML cell lines and the CD34+CD38- population was obtained. The expression of leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) and forkhead box M1 (FOXM1) was evaluated in LSCs, and the effects of GAA on malignancies and mitochondrial function were measured. LRPPRC was found to be upregulated, and GAA inhibited cell proliferation by degrading LRPPRC. GAA induced LRPPRC degradation and inhibited the activation of interleukin 6 (IL-6)/janus kinase (JAK) 1/signal transducer and activator of transcription (STAT) 3 signaling, enhancing chemosensitivity in LSCs against conventional chemotherapies, including L-Asparaginase, Dexamethasone, and cytarabine. GAA was also found to downregulate FOXM1 indirectly by regulating LRPPRC. Furthermore, GAA induced reactive oxygen species accumulation, disturbed mitochondrial homeostasis, and caused mitochondrial dysfunction. By inhibiting IL-6/JAK1/STAT3 signaling via degrading LRPPRC, GAA resulted in the elimination of LSCs. Meanwhile, GAA induced oxidative stress and subsequent cell damage by causing mitochondrial damage. Taken together, the results indicate that GAA might overcome the BMM protective effect and be considered as a novel and effective combination therapy for AML.

Gossypol Acetic Acid alleviates the Ferroptosis of Chondrocytes in Osteoarthritis by Inhibiting GPX4 Methylation.

Osteoarthritis (OA) is a chronic joint disease, usually accompanied by degeneration of the articular cartilage, fibrosis, bone hyperplasia around the joint, and damage to the entire articular surface. Gossypol is a natural phenolic compound isolated from the seed of cotton plants, and gossypol acetic acid (GAA) is a medicinal form of Gossypol. Recently, various biological activities of GAA, including anti-inflammatory and anti-tumor effects, have been widely reported. However, its effect on chondrocytes in OA has yet to be determined. In this study, we investigated the effect of GAA on ferroptosis in OA chondrocytes. The effect of GAA on the cell viability and cytotoxicity of chondrocytes in rat cells was investigated using CCK8. Western blotting, Reverse-transcription PCR (RT-PCR), and immunofluorescence staining were used to elucidate the molecular mechanisms and signaling pathways of GAA inhibition of ferroptosis in OA chondrocytes. The effect of GAA on reactive oxygen species (ROS) production and lipid peroxidation levels in chondrocytes was examined using dihydroethidium (DHE) staining and fluorescent dye BODIPY581/591 C11. in vivo, micro-CT imaging, hematoxylin and eosin staining, Safranin O-Fast staining, and immunohistochemistry were performed to evaluate the effects of GAA on OA cartilage. The results showed that GAA treatment regulated the expression of chondrocyte extracellular matrix (ECM) related factors, including ADAMTS5, MMP13, SOX9, Aggrecan, and COL1A2 and reduced the ROS and lipid peroxidation levels. Besides, Erastin could reverse the effects of GAA on chondrocytes. Similar to GAA, 5-AZA caused the reduction of ROS and lipid peroxidation levels and reversed the effect of IL-1β on the expression of ECM-related factors in OA chondrocytes. The above results clarified that GAA alleviated the ferroptosis of chondrocytes in OA by inhibiting GPX4 methylation. Our findings revealed that GAA might be developed as a drug for treating OA clinically.

APPLICATION OF MATHEMATICAL MODELING AND PHYSICO-CHEMICAL ANALYSIS METHODS IN THE PREDICTION OF BIOLOGICAL ACTIVITY AND QUALITY CONTROL OF GOSSYPOL DERIVATIVES

Objective: The purpose of this work was to evaluate in silico biological activity profiles of real and virtual molecular structures of gossypol derivatives and to develop methods of Physico-chemical analysis to control their quality. Methods: Substance of gossypol-acetic acid (GAA) and 14 virtual derivatives; PASS and ChemicDescript QSAR methods; low angle and dynamic laser light scattering (LALLS, DLS) methods; IR Spectroscopy–Cary 630 Fourier Transform IR Spectrometer, UV spectrometry–Cary-60 spectrophotometer, Optical microscopy (Altami BIO 2 microscope); Spirotox method for a sample’s biological activity. Results: A distance-based topological Balaban index (J) was successfully selected by ChemicDescript analysis; the Pa meaning by PASS Online program showed maximum (from 0.8 to 0.9) variations of antitumor and antiandrogenic and minimum of antiviral activities of GAA derivatives (Pa<0.5) despite the existing literature data. Microscopy and DLS methods demonstrated the values of high powder dispersion d=0.8 nm and weak stability of colloidal particles =-0.9 mV. According to UV data =42.4±0.8 (100 ml·g-1·cm-1) at λmax=380 nm. The LALLS method determined the GAA dissolution rate constant in ethanol: k=0.041±0.004 s-1. The calculated activation energy values of cell biosensor death process in 1 mmol solution of GAA in N,N-DMF: °bsEa=174.36±0.45 kJ·mol-1 in comparison with the solvent medium: °bsEa=213±1.55 kJ·mol-1 Conclusion: The developed approach of chemometric, laser and biotesting methods can be used for the identification of biologically active properties, as well as for qualitative analysis within the development of the standard for the pharmaceutical substance of natural polyphenols.

Targeting the miR-34a/LRPPRC/MDR1 axis collapse the chemoresistance in P53 inactive colorectal cancer.

P53 mutation is an important cause of chemoresistance in colorectal cancer (CRC). The investigation and identification of the downstream targets and underlying molecular mechanism of chemoresistance induced by P53 abnormalities are therefore of great clinical significance. In this study, we demonstrated and reported for the first time that leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is a key functional downstream factor and therapeutic target for P53 mutation-induced chemoresistance. Due to its RNA binding function, LRPPRC specifically bound to the mRNA of multidrug resistance 1 (MDR1), increasing MDR1 mRNA stability and protein expression. In normal cells, P53 induced by chemotherapy inhibited the expression of LRPPRC via miR-34a and in turn reduced the expression of MDR1. However, chemotherapy-induced P53/miR-34a/LRPPRC/MDR1 signalling pathway activation was lost when P53 was mutated. Additionally, the accumulated LRPPRC and MDR1 promoted drug resistance. Most importantly, gossypol-acetic acid (GAA) was recently reported by our team as the first specific inhibitor of LRPPRC. In CRC cells with P53 mutation, GAA effectively induced degradation of the LRPPRC protein and reduced chemoresistance. Both in vivo and in vitro experiments revealed that combination chemotherapy with GAA and 5-fluorouracil (5FU) yielded improved treatment outcomes. In this study, we reported a novel mechanism and target related to P53-induced drug resistance and provided corresponding interventional strategies for the precision treatment of CRC.

Systematic Review of Gossypol/AT-101 in Cancer Clinical Trials.

The potential of gossypol and of its R-(−)-enantiomer (R-(−)-gossypol acetic acid, AT-101), has been evaluated for treatment of cancer as an independent agent and in combination with standard chemo-radiation-therapies, respectively. This review assesses the evidence for safety and clinical effectiveness of oral gossypol/AT-101 in treating various types of cancer. The databases PubMed, MEDLINE, Cochrane, and ClinicalTrials.gov were examined. Phase I and II trials as well as single arm and randomized trials were included in this review. Results were screened to determine if they met inclusion criteria and then summarized using a narrative approach. A total of 17 trials involving 759 patients met the inclusion criteria. Overall, orally applied gossypol/AT-101 at low doses (30 mg daily or lower) was determined as well tolerable either as monotherapy or in combination with chemo-radiation. Adverse events should be strictly monitored and were successfully managed by dose-reduction or treating symptoms. There are four randomized trials, two performed in patients with advanced non-small cell lung cancer, one in subjects with head and neck cancer, and one in patients with metastatic castration-resistant prostate cancer. Thereby, standard chemotherapy (either docetaxel (two trials) or docetaxel plus cisplatin or docetaxel plus prednisone) was tested with and without AT-101. Within these trials, a potential benefit was observed in high-risk patients or in some patients with prolongation in progression-free survival or in overall survival. Strikingly, the most recent clinical trial combined low dose AT-101 with docetaxel, fluorouracil, and radiation, achieving complete responses in 11 of 13 patients with gastroesophageal carcinoma (median duration of 12 months) and a median progression-free survival of 52 months. The promising results shown in subsets of patients supports the need of further specification of AT-101 sensitive cancers as well as for the establishment of effective AT-101-based therapy. In addition, the lowest recommended dose of gossypol and its precise toxicity profile need to be confirmed in further studies. Randomized placebo-controlled trials should be performed to validate these data in large cohorts.

Gossypol Acetic Acid Attenuates Cardiac Ischemia/Reperfusion Injury in Rats via an Antiferroptotic Mechanism.

Myocardial ischemia/reperfusion (I/R) injury has been associated with ferroptosis, which is characterized by an iron-dependent accumulation of lipid peroxide to lethal levels. Gossypol acetic acid (GAA), a natural product taken from the seeds of cotton plants, prevents oxidative stress. However, the effects of GAA on myocardial I/R-induced ferroptosis remain unclear. This study investigated the ability of GAA to attenuate I/R-induced ferroptosis in cardiomyocytes along with the underlying mechanisms in a well-established rat model of myocardial I/R and isolated neonatal rat cardiomyocytes. H9c2 cells and cardiomyocytes were treated with the ferroptosis inducers erastin, RSL3, and Fe-SP. GAA could protect H9c2 cells against ferroptotic cell death caused by these ferroptosis inducers by decreasing the production of malondialdehyde and reactive oxygen species, chelating iron content, and downregulating mRNA levels of Ptgs2. GAA could prevent oxygen-glucose deprivation/reperfusion-induced cell death and lipid peroxidation in the cardiomyocytes. Moreover, GAA significantly attenuated myocardial infarct size, reduced lipid peroxidation, decreased the mRNA levels of the ferroptosis markers Ptgs2 and Acsl4, decreased the protein levels of ACSL4 and NRF2, and increased the protein levels of GPX4 in I/R-induced ex vivo rat hearts. Thus, GAA may play a cytoprotectant role in ferroptosis-induced cardiomyocyte death and myocardial I/R-induced ferroptotic cell death.

Effects of Low- and High-Level Gossypol and Sodium Butyrate Supplementation Under High-Level Gossypol Condition on Growth Performance and Intestinal Health of Hybrid Grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂)

The supplementation of gossypol in excess is noted to cause detrimental effects such as the reduction of antioxidant enzymes and disruption of lipid metabolism in animals. Studies regarding the effects of different levels of gossypol are very rare; thus, this study was conducted to evaluate the effects of low and high dietary levels of gossypol and of supplementation with 0.13 % sodium butyrate (NaB) under high gossypol conditions on the growth performance and intestinal health of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂). Four treatments were used: Feed containing 40% fish meal was used as the control group [fishmeal (FM)], the FM diet plus 0.03% gossypol acetic acid (abbreviated as gossypol) as the low-level gossypol group (gL), FM + 0.15% gossypol was used as the high-level gossypol group (gH), and FM+0.15 % gossypol with 0.13 % NaB as the repair group (gHNaB). All diets were isonitrogenous and isolipidic. The results showed that the gL treatment significantly increased specific growth rate (SGR) and feed utilization; upregulated mRNA levels of distal intestinal transforming growth factor-β1 (tgfβ1), jam, occludin, claudin3, and zo1; and downregulated mRNA levels of il8, ifnγ, and akt. The gH treatment significantly reduced SGR and feed utilization; increased distal intestinal total nitric oxide synthase (NOS) activity and nitric oxide (NO) content; upregulated mRNA levels of distal intestinal tnfα, il1β, il6, ifnγ, caspase2, caspase9, and akt; and downregulated mRNA levels of tgfβ1, jam, and zo1. NaB supplementation significantly increased distal intestinal total NOS activity and NO content; downregulated distal intestinal tnfα, il1β, ifnγ, pi3k p85, and akt mRNA levels; and increased distal intestinal tgfβ1, jam, occludin, and zo1 mRNA levels. Above all, low- and high-level gossypol exhibited positive and negative effects on growth performance, distal intestinal anti-inflammatory capacity, and tight junctions, respectively, in hybrid groupers. NaB supplementation improved distal intestinal anti-inflammatory capacity and tight junctions in hybrid groupers to a certain extent.

Investigation of Inhibition Effect of Gossypol-Acetic Acid on Gastric Cancer Cells Based on a Network Pharmacology Approach and Experimental Validation.

BackgroundGastric cancer (GC) is one of the major public health problems worldwide with high morbidity and mortality. Nowadays, traditional medicine may hold promise for the treatment of cancers. Gossypol-acetic acid (GAA) is a male contraceptive agent that shows anti-tumor effects on multiple types of cancers. However, whether GAA would inhibit the progression of GC remained unclear.MethodsThe potential targets of GAA were predicted by the Pharmmapper software and GC-related genes were obtained from the GeneCard database. The “GC-targets-GAA” network was constructed using the Cytoscape software. The PPI analysis of intersection genes was performed using the String software. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the DAVID software to explore the potential mechanism underlying the regulatory role of GAA in GC. The MTS test, plate cloning test, cell cycle and apoptosis assays were used to verify the function of GAA in GC.ResultsTen hub genes related to cell cycle progression and apoptosis were identified. Many cancer-related signaling pathways were visualized by the Cytoscape software. Among them, the PI3K-Akt signaling pathway was the highest-ranked pathway. The MTS test and plate cloning test showed that GAA inhibited the proliferation of GC cells. The cell cycle and apoptosis assays showed that GAA induced G1 phase cell cycle arrest and apoptosis in GC cells.ConclusionOur study demonstrated the anti-tumor effect of GAA on GC through multiple targets and signaling pathways. These results provided a theoretical basis for further investigation of GAA in preclinical and clinical studies, and suggested the potential use of GAA as a novel therapeutic agent for the treatment of GC.

Proteasome-Independent Protein Knockdown by Small-Molecule Inhibitor for the Undruggable Lung Adenocarcinoma.

Therapeutic target identification and corresponding drug development is a demanding task for the treatment of lung adenocarcinoma, especially the most malignant proximal-proliferative subtype without druggable protein kinase mutations. Using a cell-SELEX-generated aptamer, we discovered a new tumor driver protein, leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), which is specifically overexpressed in the most lethal subtype of lung adenocarcinoma. Targeted LRPPRC protein knockdown is a promising therapeutic strategy for the undruggable LUAD (lung adenocarcinoma). Nevertheless, LRPPRC is mainly located in mitochondria and degraded by protease. Current protein knockdown approaches, such as proteolysis-targeting chimeras (PROTACs), have limitations in their applications to the proteins degraded through proteasome-independent ways. Here, we designed an aptamer-assisted high-throughput method to screen small molecules that could bind to LRPPRC directly, disrupt the interaction of LRPPRC with its stabilizing chaperon protein, and lead to LRPPRC degradation by mitochondrial protease. The screened compound, gossypolacetic acid (GAA), is an old medicine that can accomplish the new function for targeted LRPPRC knockdown. It showed significant antitumor effects even with the LRPPRC-positive patient-derived tumor xenograft (PDX) model. This work not only extended the application of aptamers to screen small-molecule inhibitors for the undruggable lung cancers, but more importantly provided a new strategy to develop protein knockdown methods beyond the proteasome system.

Cytokine-regulating activity of anti-virus preparation CelAgripus in Burkitt's lymphoma stable B-cell lines.

Cytokines activated in response to immunosuppressive viral infections can directly or indirectly affect the neoplastic transformation of B cells. In this study, we studied a new substance designed to produce the antiviral drug CelAgrip (CA, CelAgripus), which exhibits interferon (IFN) and cytokine-inducing activity and, apparently, can be used as an activator of antiviral immunity. Purpose - is to evaluate the cytokine-regulating effect of CA in Burkitt's lymphoma (LB) cell lines latently infected with the Epstein-Barr virus (EBV). to study the CA-induced expression of the cytokine genes IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IFN-α, IFN -γ, IFN-β, IFN-λ1, IFN-λ2, IFN-λ3, TNF-α in normal and EBV transformed LB cells. Cell line: the human embryo fibroblasts (HEF), Namalva, Daudi, Raji, P3HR-1. Preparations: CA, gossypol-acetic acid (GAA), sodium carboxymethyl cellulose (Na-CMC). RT-PCR and methods for assessing cytotoxicity (MTT and Scepter 2.0 Merck cell counter). The effect of the CA preparation on the expression of IFN-λ, IL-1β, IL-6, IL-8 and IL-10 genes was revealed. We observed the activation of gene expression of IFN-λ, IL-1β, IL-6, IL-8 and suppression of IL-10 gene activity when treatment CA of LB cells. The substance CA has new effects on the activation of the expression of a number of key cytokine genes in stable Burkitt lymphoma cell lines.

Dietary gossypol reduced intestinal immunity and aggravated inflammation in on-growing grass carp (Ctenopharyngodon idella)

The present study explored the effects of dietary gossypol on the gut health of on-growing grass carp. The fish were fed six diets containing different levels of free gossypol (0, 121.38, 243.94, 363.89, 759.93 and 1162.06 mg/kg diet) from gossypol-acetic acid for 60 days and then challenged with Aeromonas hydrophila for 14 days. The results showed that dietary gossypol (1) could aggravate enteritis and damage the structure of intestinal epithelial cells, (2) decreased the lysozyme (LZ) and Acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and it down-regulated the Hepcidin (rather than distal intestine (DI)), immunoglobulin Z (IgZ), liver-expressed antimicrobial peptide (LEAP)-2B, Mucin2 and β-defensin-1 mRNA levels in the proximal intestine (PI), mid intestine (MI) and DI, (3) up-regulated intestinal pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interferon γ2 (IFN-γ2), interleukin 1β (IL-1β), IL-6 (only in PI), IL-8 and IL-12p35 mRNA levels partly related to nuclear factor kappa B (NF-κB) signalling, and (4) down-regulated the mRNA levels of anti-inflammatory cytokines such as transforming growth factor (TGF)-β1, TGF-β2, interleukin 4/13A (IL-4/13A) (except IL-4/13B), IL-10 and IL-11 partly relating to target of rapamycin (TOR) signalling in the intestines of on-growing grass carp. Moreover, the dietary gossypol had no impact on the LEAP-2A, IL-12P40, IL-17D, IL-10, NF-κBp52, IKKα and eIF4E-binding proteins 2 (4E-BP2) mRNA levels in the intestines. Finally, based on the intestinal histopathological results, enteritis morbidity, LZ activity and IgM content, the safe dose of gossypol in the diets for on-growing grass carp should be less than 103.42 mg/kg diet.

Gossypol reduced the intestinal amino acid absorption capacity of young grass carp (Ctenopharyngodon idella)

This study was conducted to evaluate the effects of gossypol on the growth, intestinal histology, intestinal amino acid (AA) absorption capacity and the safe upper limit of gossypol in the diets for young grass carp (Ctenopharyngodon idella) growth. The fish were fed six diets containing different levels of free gossypol (0, 125 (121.38), 250 (243.94), 375 (363.89), 775 (759.93) and 1175 (1162.06) mg kg−1 diet) from gossypol-acetic acid for 60 days. Results indicated that gossypol could reduce the growth performance and damage the intestines of young grass carp. Interestingly, gossypol modulated the absorptive abilities of various AAs, and mechanisms are not unified in fish. Firstly, gossypol decreased the contents of tryptophan (Trp), threonine (Thr), valine (Val), phenylalanine (Phe), glycine (Gly), serine (Ser) and tyrosine (Tyr) in the three intestinal segments of fish associated with down-regulating the mRNA levels of solute carrier (SLC) 6A19b, SLC7A5, SLC7A8, SLC1A5, SLC38A2 and peptide transporter 1 (PepT1) in the intestine. Secondly, gossypol reduced the contents of methionine (Met), isoleucine (Ile), proline (Pro), cysteine (Cys) and arginine (Arg) in the proximal intestine (PI) and mid intestine (MI) of fish by down-regulating the mRNA levels of SLC7A7, SLC7A6, SLC7A1, SLC6A19b, SLC7A5, SLC7A8, SLC1A5, SLC38A2 and PepT1 in the PI and MI. Thirdly, gossypol inhibited the aspartic acid (Asp) and taurine (Tau) contents in the PI and MI of fish by down-regulating the mRNA levels of SLC1A2a, SLC1A3, PepT1 and SLC6A6 in the PI and MI. Fourthly, gossypol suppressed the lysine(Lys) and histidine (His) contents in the MI and distal intestine (DI) of fish due to down-regulating the mRNA levels of SLC7A7, SLC7A5 and SLC38A2 in the MI, and SLC7A6, SLC7A1, SLC7A8 and PepT1 in the MI and DI. Gossypol down-regulated the above AA and peptide transporters in the intestine of fish partly related to suppressing the target of rapamycin (TOR) signalling pathway. Based on percent weight gain (PWG) and feed efficiency (FE), the safe upper limit of gossypol in the diets for young grass carp growth were estimated to be 182 and 179 mg kg−1 diet, respectively. In summary, gossypol reduced the intestinal AA absorption capacity of young grass carp partly relate to damaging the intestinal structure, down-regulating the mRNA levels of AA and peptide transporters, and inhibiting the TOR signalling pathway.

Measurement and correlation of the solubility of gossypol acetic acid and gossypol acetic acid of optical activity in different solvents

The equilibrium method was used to measure the solubility of gossypol acetic acid and gossypol acetic acid of optical activity in isopropyl alcohol, ethanol, acetic acid and ethyl acetate at temperature from 288.15 to 315.15. The Empirical equation and the Apelblat equation model were adopted to correlate the experimental data. For gossypol acetic acid, the root-mean-square deviations (RMSD) were observed in the range of 0.023-4.979 and 0.0112-0.614 for the Empirical equation and the Apelblat equation, respectively. For gossypol acetic acid of optical activity, the RMSD were observed in the range of 0.021-2.211 and 0.021-2.243 for the Empirical equation and the Apelblat equation, individually. And the maximum relative average deviation was 7.5%. Both equations offered an accurate mathematical expression of the experimental results. The calculated solubility showed a good relationship with the experimental solubility for most of solvents. This study provided valuable datas not only for optimizing the process of purification of gossypol acetic acid of optical activity in industry but also for further theoretical studies.

Identification of natural products and their derivatives as promising inhibitors of protein glycation with non-toxic nature against mouse fibroblast 3T3 cells

<p>This study was performed to identify new inhibitors of protein glycation <em>in vitro</em>. Protein glycation is one of the major causes of late diabetic complications. In this study, terpenoids and alkaloids, isolated from different medicinal plants, along with their derivatives, were evaluated for their antiglycation activity <em>in vitro,</em> while MTT assay on mouse fibroblast 3T3 cells was used to assess their potential cytotoxicity. Among the tested compounds, gossypol (2,2′-<em>bis</em>-(formyl-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene) (<strong>1</strong>), isolated from<em> Gossypium herbaceum, </em>and its derivatives,<em> </em>gossypol acetic acid (<strong>2</strong>), gossypolidene- 4-aminoantipyrine (<strong>4</strong>), and gazolidone (<strong>6</strong>), showed a potent antiglycation activity (IC<sub>50</sub> < 16 <em>µ</em>M), while gossypolidene-4-aminoantipyrine (<strong>5</strong>) showed a significant antiglycation activity with IC<sub>50 </sub>value 82.934±2.924<em> µ</em>M, in BSA-fluorescence assay. Alkaloid, noscapine (3S)-6,7-Dimethoxy-3-[(5R)-4-methoxy-6-methyl-5,6,7,8-tetrahy-dro-1,3-dioxolo[4,5-g]isoquinolin-5-yl] isobenzofuran-1(3<em>H</em>)-one (<strong>7</strong>), isolated from <em>Papaver somniferum, N</em>-nitrosoaphyllinic acid (<strong>9</strong>), a derivative of alkaloid aphylline<em>, </em>and 2<em>H</em>-quinolizine, octahydro salt (<strong>11</strong>), a salt of alkaloid lupinine, exhibited significant inhibition activity with<em> </em>IC<sub>50 </sub>values 152.662±5.432, 393.758 ±4.001 µM and 110.203±4.816µM, respectively. Similarly, compounds<strong> </strong>gossypolidene thiocarbamide (<strong>3</strong>), deoxypeganine hydrochloride (<strong>8</strong>)<strong>, </strong>lupinine (<strong>10</strong>) and cytisine (<strong>12</strong>) showed moderate inhibition with IC<sub>50</sub> values of 401.865 ±18.450, 863.322 ±6.415, 712.176±7.745, and 728.462±2.331<em> </em>µM, respectively. The results were compared with the standard antiglycation agent, rutin (<strong>13</strong>) (IC<sub>50 </sub>=98.012±2.030 µM).</p>Cellular cytotoxicity assay showed only gossypol acetic acid (<strong>2</strong>) and gossypolidene thiocarbamide (<strong>3</strong>) as somewhat toxic to 3T3 (mouse fibroblast) cells with IC<sub>50 </sub>values<em> </em>2.07 ±0.61 and 5.00 ±1.89 µM, respectively. Cycloheximide was used as a standard in this assay with IC<sub>50</sub> value 0.3 ± 0.089 μM

Toxicity of Gossypol from Cottonseed Cake to Sheep Ovarian Follicles.

Gossypol, a polyphenol compound produced by cotton plant, has proven reproductive toxicity, but the effects of gossypol on sheep ovaries are unknown. This study was aimed to determine the in vitro and in vivo effects of gossypol on the ovarian follicles of sheep. This trial was divided into two experiments. In the first one, we used twelve non-pregnant, nulliparous, Santa Inês crossbred ewes, which were randomly distributed into two equal groups and fed diets with and without cottonseed cake. Feed was offered at 1.5% of the animal’s body weight for 63 days. The concentrations of total and free gossypol in the cottonseed cake were 3.28 mg/g and 0.11 mg/g, respectively. Throughout the trial period, no animal showed clinical signs of toxicity and no effects on body weight were observed. However, there was a significantly lower number of viable ovarian follicles (20.6%) and higher number of atretic follicles (79.4%) in the gossypol-fed sheep compared to the control (85.1 and 34.9%, respectively). These findings were observed at all stages of follicular development. In the second experiment, eight ovaries from slaughterhouse were cultured with different concentrations of gossypol acetic acid (0, 5, 10 and 20 μg/mL) for 24 hours or seven days. The in vitro action of gossypol resulted in a significant decrease in viable ovarian follicles, especially the primary and transition follicles, and a significant increase in the number of atretic follicles after 24 hours of culture. These follicles were greatly affected when cultured with gossypol for seven days. It is concluded that gossypol present in cotton seeds directly acts on ovarian follicles in sheep to increase atresia.

Gossypol Acetic Acid Prevents Oxidative Stress-Induced Retinal Pigment Epithelial Necrosis by Regulating the FoxO3/Sestrin2 Pathway.

The late stage of dry age-related macular degeneration (AMD), or geographic atrophy (GA), is characterized by extensive retinal pigment epithelial (RPE) cell death, and a cure is not available currently. We have recently demonstrated that RPE cells die from necrosis in response to oxidative stress, providing a potential novel mechanism for RPE death in AMD. In this study, we screened U.S. Food and Drug Administration-approved natural compounds and identified gossypol acetic acid (GAA) as a potent inhibitor of oxidative stress-induced RPE cell death. GAA induces antioxidative response and inhibits accumulation of excessive reactive oxygen species in cells, through which it prevents the activation of intrinsic necrotic pathway in response to oxidative stress. Sestrin2 (SESN2) is found to mediate GAA function in antioxidative response and RPE survival upon oxidative stress. Moreover, Forkhead box O3 transcription factor (FoxO3) is further found to be required for GAA-mediated SESN2 expression and RPE survival. Mechanistically, GAA promotes FoxO3 nuclear translocation and binding to the SESN2 enhancer, which in turn increases its transcriptional activity. Taken together, we have identified GAA as a potent inhibitor of oxidative stress-induced RPE necrosis by regulating the FoxO3/SESN2 pathway. This study may have significant implications in the therapeutics of age-related diseases, especially GA.

Influence of gossypol acetic acid on the growth of human adenoid cystic carcinoma ACC-M cells and the expression of DNA methyltransferase 1.

We investigated the effects of gossypol acetic acid (GAA) on the proliferation, apoptosis, and expression of DNA methyltransferase 1 (DNMT1) mRNA in human adenoid cystic carcinoma (ACC-M) cells in vitro. The proliferation and apoptosis of ACC-M cells after treatment with different concentrations of GAA were detected using Cell Counting Kit-8 and flow cytometry, respectively. DNMT1 mRNA expression was measured by real-time fluorescence quantitative polymerase chain reaction. The growth of ACC-M cells was inhibited after treatment with GAA for 24, 48, and 72 h. The apoptotic rates of ACC-M cells after treatment with GAA for 72 h were higher than those of control cells (without treatment) (P < 0.05). DNMT1 mRNA expression in ACC-M after treatment with GAA for 72 h was lower than that in control cells (P < 0.05). GAA had inhibitory effects on the proliferation and induced apoptosis of human ACC-M cells, while GAA also reduced the expression level of DNMT1 mRNA in ACC-M cells.

Induction of H2AX phosphorylation in tumor cells by gossypol acetic acid is mediated by phosphatidylinositol 3-kinase (PI3K) family.

BackgroundH2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including Ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. Our study shows that gossypol acetic acid (GAA) alone can induce γH2AX in Human mucoepidermoid carcinoma cell line (MEC-1) in vitro. Thus, we further examined the possible mechanisms of GAA to induce γH2AX in tumor cells.Materials and methodsThe PI3K inhibitors caffeine and wortmannin were used in an effort to identify the kinase(s) responsible for GAA -induced γH2AX in MEC-1 cells. DNA dependent protein kinase (DNA-PK) - proficient and –deficient cells, human glioma cell lines M059K and M059J, were also used to evaluate the kinases responsible for GAA induced H2AX phosphorylation. γH2AX expression was detected by immunofluorescent microscopy. Flow cytometry assay was used to assay γH2AX and cell cycle.ResultsGAA induced H2AX phosphorylation in a cell cycle-dependent manner and a significant G0/G1 phase arrest in MEC-1 cells was shown. Caffeine and wortmannin significantly inhibited GAA-induced H2AX phosphorylation in MEC-1 cells. GAA induced H2AX phosphorylation in M059K, but not in M059J. Taken together, these data suggested that GAA treatment alone could induce H2AX phosphorylation in a cell cycle dependent manner in MEC-1 and M059K, but not in M059J cells. A significant G0/G1 phase arrest was shown in MEC-1.ConclusionsThe member of PI3K family, DNA-PK, ATM and ATR are involved in the H2AX phosphorylation of MEC-1 cells.

RhoA/ROCK/PTEN signaling is involved in AT-101-mediated apoptosis in human leukemia cells in vitro and in vivo

R-(−)-gossypol acetic acid (AT-101) is a natural cottonseed product that exhibits anticancer activity. However, the molecular mechanism behind the antileukemic activity of AT-101 has not been well characterized. In this study, we investigated how AT-101 induces apoptosis in human leukemia cells. Exposure to AT-101 significantly increased apoptosis in both human leukemia cell lines and primary human leukemia cells. This increase was accompanied by the activation of caspases, cytochrome c release, Bcl2-associated X protein (Bax) translocation, myeloid cell leukemia-1 (Mcl-1) downregulation, Bcl-2-associated death promoter (Bad) dephosphorylation, Akt inactivation, and RhoA/Rho-associated coiled-coil containing protein kinase 1/phosphatase and tensin homolog (RhoA/ROCK1/PTEN) activation. RhoA, rather than caspase-3 cleavage, mediated the cleavage/activation of ROCK1 that AT-101 induced. Inhibiting RhoA and ROCK1 activation by C3 exoenzyme (C3) and Y27632, respectively, attenuated the ROCK1 cleavage/activation, PTEN activity, Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and apoptosis mediated by AT-101. Knocking down ROCK1 expression using a ROCK1-specific siRNA also significantly abrogated AT-101-mediated apoptosis. Constitutively active Akt prevented the AT-101-induced Mcl-1 downregulation, Bad dephosphorylation, and apoptosis. Conversely, AT-101 lethality was potentiated by the phosphatidylinositol 3-kinase inhibitor LY294002. In vivo, the tumor growth inhibition caused by AT-101 was also associated with RhoA/ROCK1/PTEN activation and Akt inactivation in a mouse leukemia xenograft model. Collectively, these findings suggest that AT-101 may preferentially induce apoptosis in leukemia cells by interrupting the RhoA/ROCK1/PTEN pathway, leading to Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and Bax translocation, which culminate in mitochondrial injury and apoptosis.