ABSTRACT
MicroRNAs (miRNAs) have been demonstrated to act as crucial modulators with considerable impacts on the immune system. Cottonseed meal is often used as a protein source in aqua feed, cottonseed meal contains gossypol, which is harmful to animals. However, there is a lack of research on the role of miRNAs in fish exposed to gossypol stress. To determine the regulatory effects of miRNAs on gossypol toxicity, Cyprinus carpio were given to oral administration of 20 mg/kg gossypol for 7 days, and the gossypol concentration in the tissues was tested. Then, we detected spleen index, histology, immune enzyme activities of fish induced by gossypol. The results of miRNA sequencing revealed 8 differentially expressed miRNAs in gossypol group, and miR-214_L-1R+4 was found involved in immune response induced by gossypol. The potential targets of miR-214_L-1R+4 were predicted, and found a putative miR-214_L-1R+4 binding site in the 3'UTR of MyD88a. Furthermore, dual-luciferase reporter assays displayed miR-214_L-1R+4 decreased MyD88a expression through binding to the 3'UTR of MyD88a. Moreover, miR-214_L-1R+4 antagomir were intraperitoneally administered to C. carpio, down-regulated miR-214_L-1R+4 could increase MyD88a expression, as well as inflammatory cytokines and anti-inflammatory cytokines expression. These findings revealed that miR-214_L-1R+4 via the MyD88-dependent signaling pathway modulate the immune response to gossypol in C. carpio spleen.
Subject(s)
Carps , Fish Proteins , Gossypol , MicroRNAs , Myeloid Differentiation Factor 88 , Signal Transduction , Animals , Carps/immunology , Carps/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Gossypol/pharmacology , Gossypol/administration & dosage , Signal Transduction/drug effects , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/metabolism , Immunity, Innate/drug effects , Immunity, Innate/geneticsABSTRACT
Gossypol, a naturally occurring compound found in cottonseed meal, shows promising therapeutic potential for human diseases. However, within the aquaculture industry, it is considered an antinutritional factor. The incorporation of cottonseed meal into fish feed introduces gossypol, which induces intracellular stresses and hinders overall health of farmed fish. The aim of this study is to determine the role of General control nonderepressible 2 (gcn2), a sensor for intracellular stresses in gossypol-induced stress responses in fish. In the present study, we established two gcn2 knockout zebrafish lines. A feeding trial was conducted to assess the growth-inhibitory effect of gossypol in both wild type and gcn2 knockout zebrafish. The results showed that in the absence of gcn2, zebrafish exhibited increased oxidative stress and apoptosis when exposed to gossypol, resulting in higher mortality rates. In feeding trial, dietary gossypol intensified liver inflammation in gcn2-/- zebrafish, diminishing their growth and feed conversion. Remarkably, administering the antioxidant N-acetylcysteine (NAC) was effective in reversing the gossypol induced oxidative stress and apoptosis, thereby increasing the gossypol tolerance of gcn2-/- zebrafish. Exposure to gossypol induces more severe mitochondrial stress in gcn2-/- zebrafish, thereby inducing metabolic disorders. These results reveal that gcn2 plays a protective role in reducing gossypol-induced oxidative stress and apoptosis, attenuating inflammation responses, and enhancing the survivability of zebrafish in gossypol-challenged conditions. Therefore, maintaining appropriate activation of Gcn2 may be beneficial for fish fed diets containing gossypol.
Subject(s)
Apoptosis , Gossypol , Inflammation , Oxidative Stress , Zebrafish , Animals , Gossypol/toxicity , Gossypol/pharmacology , Gossypol/administration & dosage , Oxidative Stress/drug effects , Apoptosis/drug effects , Inflammation/chemically induced , Animal Feed/analysis , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Diet/veterinary , Fish Diseases/chemically induced , Fish Diseases/immunology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
The aim of this study was to evaluate if the cottonseed intake during gestation and lactation affects the ovarian population in ewes and lambs. Therefore, 39 ewes were evaluated during 10 months under two treatments: Cottonseed and soybeans. The quantification of ovarian follicular dynamics was analyzed by ultrasound and the determination of progesterone and estradiol levels was interpreted by radioimmunoassay. After weaning, ovaries of lambs (n = 10) were collected by ovariectomy and fixed for the assessment of follicular parameters as normality, classification, diameter, ultrastructure, stereology, and as well as immunoexpression of the α-estradiol receptor α (ER-α). The results showed that the cottonseed consumption altered neither the ovarian nor the hormonal follicular dynamics of Santa Inês ewes after calving and did not affect the normality, classification, diameter, stereology and follicular ultrastructure of offspring. Nevertheless, the offspring of ewes fed with cottonseed showed high ER-α immunoexpression in the ovarian structures. It is concluded that cottonseed did not affect the maternal-descendant follicular dynamics. However, lambs' ovaries had highest α-ER immunoexpression in oocytes, granulosa and theca cells and corpus luteum. This fact warns of a possible change in the future steroidogenic response of these lambs that had progenitors consuming cottonseed in their reproductive period.
Subject(s)
Gossypol/pharmacology , Ovary/drug effects , Sheep , Animals , Female , Gossypol/administration & dosage , Lactation , Oocytes/metabolism , Pregnancy , Progesterone , ReproductionABSTRACT
Metastasis is the most prevalent cause of cancer-associated deaths amongst patients with cervical cancer. Epithelial-mesenchymal transition (EMT) is essential for carcinogenesis, and it confers metastatic properties to cancer cells. Gossypol is a natural polyphenolic compound with anti-inflammation, anti-oxidant, and anticancer activities. In this study, we investigated the antimetastatic and antitumour effects of gossypol on human cervical cancer cells (HeLa and SiHa cells). Gossypol exerted a strong inhibition effect on the migration and invasion of human cervical cancer cells. It reduced the focal adhesion kinase (FAK) pathway-mediated expression of matrix metalloproteinase-2 and urokinase-type plasminogen activator, subsequently inhibiting the invasion of SiHa cells. In addition, gossypol reversed EMT induced by transforming growth factor beta 1 (TGF-[Formula: see text]1) and up-regulated epithelial markers, such as E-cadherin but significantly suppressed Ras homolog family member (Rho)A, RhoB, and p-Samd3. The tail vein injection model showed that gossypol treatment via oral gavage reduced lung metastasis. Gossypol also decreased tumour growth in vivo in the nude mouse xenograft model. All these findings suggest that gossypol suppressed the invasion and migration of human cervical cancer cells by targeting the FAK signaling pathway and reversing TGF-[Formula: see text]1-induced EMT. Hence, gossypol warrants further attention for basic mechanistic studies and drug development.
Subject(s)
Antineoplastic Agents, Phytogenic , Epithelial-Mesenchymal Transition , Gossypol/pharmacology , Gossypol/therapeutic use , Neoplasm Metastasis/prevention & control , Peptide Hydrolases/metabolism , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/etiology , Animals , Cell Movement/drug effects , Disease Models, Animal , Epithelial-Mesenchymal Transition/drug effects , Female , Gossypol/administration & dosage , HeLa Cells , Heterografts , Humans , Mice, Nude , Neoplasm Invasiveness , Neoplasm Transplantation , Phytotherapy , Uterine Cervical Neoplasms/pathologyABSTRACT
INTRODUCTION: AT101, the R-(-)-enantiomer of the cottonseed-derived polyphenol gossypol, is a promising drug in glioblastoma multiforme (GBM) therapy due to its ability to trigger autophagic cell death but also to facilitate apoptosis in tumor cells. It does have some limitations such as poor solubility in water-based media and consequent low bioavailability, which affect its response rate during treatment. To overcome this drawback and to improve the anti-cancer potential of AT101, the use of cubosome-based formulation for AT101 drug delivery has been proposed. This is the first report on the use of cubosomes as AT101 drug carriers in GBM cells. MATERIALS AND METHODS: Cubosomes loaded with AT101 were prepared from glyceryl monooleate (GMO) and the surfactant Pluronic F-127 using the top-down approach. The drug was introduced into the lipid prior to dispersion. Prepared formulations were then subjected to complex physicochemical and biological characterization. RESULTS: Formulations of AT101-loaded cubosomes were highly stable colloids with a high drug entrapment efficiency (97.7%) and a continuous, sustained drug release approaching 35% over 72 h. Using selective and sensitive NMR diffusometry, the drug was shown to be efficiently bound to the lipid-based cubosomes. In vitro imaging studies showed the high efficiency of cubosomal nanoparticles uptake into GBM cells, as well as their marked ability to penetrate into tumor spheroids. Treatment of GBM cells with the AT101-loaded cubosomes, but not with the free drug, induced cytoskeletal rearrangement and shortening of actin fibers. The prepared nanoparticles revealed stronger in vitro cytotoxic effects against GBM cells (A172 and LN229 cell lines), than against normal brain cells (SVGA and HMC3 cell lines). CONCLUSION: The results indicate that GMO-AT101 cubosome formulations are a promising basic tool for alternative approaches to GBM treatment.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Brain Neoplasms/drug therapy , Drug Carriers/chemistry , Glioblastoma/drug therapy , Gossypol/analogs & derivatives , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/pharmacokinetics , Biological Availability , Brain Neoplasms/pathology , Cell Line, Tumor , Colloids/chemistry , Colloids/pharmacology , Cytoskeleton/drug effects , Delayed-Action Preparations/chemistry , Drug Carriers/administration & dosage , Drug Carriers/pharmacokinetics , Drug Delivery Systems/methods , Glioblastoma/pathology , Glycerides/chemistry , Gossypol/administration & dosage , Gossypol/pharmacokinetics , Gossypol/pharmacology , Humans , Lipids/chemistry , Magnetic Resonance Spectroscopy/methods , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Poloxamer/chemistry , SolubilityABSTRACT
A cotonicultura tem forte fator de impacto na economia nacional, e o estado de Mato Grosso se destaca por ser o maior produtor de algodão herbáceo e deter o maior rebanho bovino do país, condições essas que estimulam o uso do caroço, da torta e do farelo de algodão na alimentação animal. Considerando que o gossipol está presente nos subprodutos do algodão e que seus efeitos sobre a reprodução podem reduzir a fertilidade dos animais, objetivou-se, com essa pesquisa, avaliar o efeito da ingestão de dietas com diferentes teores de gossipol livre por dia sobre a morfometria testicular e a qualidade seminal de touros da raça Nelore. Foram utilizados 28 touros, distribuídos aleatoriamente em seis tratamentos: T0, 0 grama de gossipol livre/touro/dia ; T1, 1,08 grama de gossipol livre/touro/dia; T2, 2,07 gramas de gossipol livre/touro/dia; T3, 3,24 gramas de gossipol livre/touro/dia; T4, 3,82 gramas de gossipol livre/touro/dia e T5, 5,08 gramas de gossipol livre/touro/dia. Os animais de cada tratamento foram mantidos confinados em área média de 100m2, dotada de bebedouro, cochos para mistura mineral e para volumoso/concentrado. O consumo de 3,24 gramas de gossipol livre/touro/dia alterou a qualidade espermática e a morfometria testicular de touros.(AU)
The cotton industry has a strong impact factor in the Brazilian economy and the state of Mato Grosso stands out for being the largest upland-type cotton producer and also holds the largest cattle herd in the country, conditions that stimulate the use of cottonseed pie and cottonseed meal in animal feed. Whereas gossypol is present in cotton by-products and their effects on reproduction can reduce the animal fertility, this research evaluated the effect of diets with different free gossypol contents in the testicular morphometry and semen quality of Nelore bulls. 28 bulls were randomly distributed in six treatments: T0, receiving no free gossypol; T1, receiving 1.08g / bull / day; T2, receiving 2.07g / bull / day; T3, receiving 3.24g / bull / day; T4, receiving 3.82g / bull / day and T5, receiving 5.08g / bull / day of free gossypol respectively. The animals in each treatment were kept confined in an averaged area of 100m2, having fresh water, troughs for mineral mixture and roughage / concentrate. The consumption of 3.24g of free gossypol / bull / day altered the bulls sperm quality and testicular morphometry.(AU)
Subject(s)
Animals , Male , Cattle , Testis/anatomy & histology , Cottonseed Oil/administration & dosage , Gossypol/administration & dosage , Semen Analysis , Infertility, Male/veterinaryABSTRACT
PURPOSE: Glioblastoma multiforme (GBM) is a poorly curable disease due to its profound chemoresistance. Despite recent advances in surgery, radiotherapy and chemotherapy, the efficient treatment of GBMs is still a clinical challenge. Beside others, AT101, the R-(-) enantiomer of gossypol, and demethoxycurcumin (DMC), a curcumin-related demethoxy compound derived from Curcuma longa, were considered as possible alternative drugs for GBM therapy. METHODS: Using different human primary GBM cell cultures in a long-term stimulation in vitro model, the cytotoxic and anti-proliferative effects of single and combined treatment with 5 µM AT101 and 5 µM or 10 µM DMC were investigated. Furthermore, western blots on pAkt and pp44/42 as well as JC-1 staining and real-time RT-PCR were performed to understand the influence of the treatment at the molecular and gene level. RESULTS: Due to enhanced anti-proliferative effects, we showed that combined therapy with both drugs was superior to a single treatment with AT101 or DMC. Here, by determination of the combination index, a synergism of the combined drugs was detectable. Phosphorylation and thereby activation of the kinases p44/42 and Akt, which are involved in proliferation and survival processes, were inhibited, the mitochondrial membrane potential of the GBM cells was altered, and genes involved in dormancy-associated processes were regulated by the combined treatment strategy. CONCLUSION: Combined treatment with different drugs might be an option to efficiently overcome chemoresistance of GBM cells in a long-term treatment strategy.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Brain Neoplasms/drug therapy , Diarylheptanoids/pharmacology , Glioblastoma/drug therapy , Gossypol/analogs & derivatives , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/pharmacology , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Proliferation/drug effects , Diarylheptanoids/administration & dosage , Dose-Response Relationship, Drug , Drug Synergism , Gene Expression/drug effects , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Gossypol/administration & dosage , Gossypol/pharmacology , Humans , Membrane Potential, Mitochondrial/drug effects , Signal Transduction/drug effects , Tumor Cells, CulturedABSTRACT
Metastasis is the most prevalent cause of treatment failure in patients with colon cancer. Gossypol is reported to exhibit antioxidant, anticancer, antivirus and antimicrobial properties. However, the effects of gossypol on cancer invasion and tumour growth of human colon cancer remain unclear. This study aimed to provide molecular evidence associated with the antimetastatic and anti-tumour effects of gossypol on human colorectal carcinoma (CRC) cells. Gossypol inhibited the viability of human colon cancer cells in a dose-dependent manner. Gossypol was sufficient to reduce the invasion, migration and adhesion in DLD-1 and COLO 205 cells. Zymography and western blot assay showed that gossypol reduced the activities and protein expression of urokinase-type plasminogen activator (u-PA), respectively. Gossypol suppressed the level of p-focal adhesion kinase (FAK) and epithelial-to-mesenchymal transition markers, including N-cadherin, fibronectin and vimentin. Gossypol also inhibited the lung metastasis of DLD-1 cells, as indicated by the nude mouse model. These results suggested that gossypol inhibited the metastatic properties of human colon cancer cells by targeting u-PA through the FAK pathway, suggesting that gossypol could be used as an adjuvant therapeutic agent for the treatment of human colon cancer cells.
Subject(s)
Colonic Neoplasms/drug therapy , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gossypol/administration & dosage , Lung Neoplasms/prevention & control , Urokinase-Type Plasminogen Activator/metabolism , Animals , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Epithelial-Mesenchymal Transition/drug effects , Fibronectins/genetics , Fibronectins/metabolism , Focal Adhesion Protein-Tyrosine Kinases/genetics , Humans , Lung Neoplasms/secondary , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Metastasis/prevention & control , Urokinase-Type Plasminogen Activator/genetics , Vimentin/genetics , Vimentin/metabolismABSTRACT
Leukemia stem cells (LSCs) are deemed to the mainspring for treatment failure in acute myeloid leukemia (AML). Conventional chemotherapeutic drugs fail to eradicate leukemia stem cells, which becomes the root of drug resistance and disease recurrence. Hence, new therapeutic strategies targeting LSCs are supposed to be critical for patients with AML. Here we report that combination of Bcl-2 inhibitor AT-101 and chemotherapeutic drug idarubicin (IDA) results in synergistic lethality in CD34+CD38- leukemia stem-like cells sorted from KG-1α and Kasumi-1 AML cell lines and primary CD34+ AML cells in vitro while sparing the normal counterparts. In addition, combinatorial treatment also significantly inhibits the growth of patient-derived xenograft (PDX) mouse models generated from FLT3-ITDmut AML patient in vivo. Mechanistically, the synergistic effects of AT-101 with IDA to induce cell death are closely associated with blockage of DNA damage repair and thus activates the intrinsic apoptotic pathway. In summary, these findings suggest that combinatorial therapy with AT-101 and IDA selectively eliminates leukemia stem-like cells both in vitro and in vivo, representing a potent and alternative salvage therapy for the treatment of relapsed and refractory patients with AML.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , DNA Repair/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Synthetic Lethal Mutations/drug effects , Adolescent , Adult , Aged , Animals , Biomarkers, Tumor/genetics , Case-Control Studies , Cell Proliferation/drug effects , DNA Repair/genetics , Drug Synergism , Female , Follow-Up Studies , Gossypol/administration & dosage , Gossypol/analogs & derivatives , Humans , Idarubicin/administration & dosage , Leukemia, Myeloid, Acute/genetics , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Prognosis , RUNX1 Translocation Partner 1 Protein/antagonists & inhibitors , RUNX1 Translocation Partner 1 Protein/genetics , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Young Adult , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
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 1162.06â¯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 ß-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 1ß (IL-1ß), 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)-ß1, TGF-ß2, 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 103.42â¯mg/kg diet.
Subject(s)
Carps , Fish Diseases/immunology , Gossypol/analogs & derivatives , Immunity, Innate/immunology , Inflammation/veterinary , Intestines/immunology , Aeromonas hydrophila/physiology , Animal Feed/analysis , Animals , Diet/veterinary , Dietary Supplements/analysis , Dose-Response Relationship, Drug , Fish Diseases/chemically induced , Fish Diseases/microbiology , Gossypol/administration & dosage , Gossypol/metabolism , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/veterinary , Immunity, Innate/drug effects , Inflammation/chemically induced , Inflammation/immunology , Intestines/drug effects , Random AllocationABSTRACT
BACKGROUND: The Musashi (MSI) family of RNA-binding proteins is best known for the role in post-transcriptional regulation of target mRNAs. Elevated MSI1 levels in a variety of human cancer are associated with up-regulation of Notch/Wnt signaling. MSI1 binds to and negatively regulates translation of Numb and APC (adenomatous polyposis coli), negative regulators of Notch and Wnt signaling respectively. METHODS: Previously, we have shown that the natural product (-)-gossypol as the first known small molecule inhibitor of MSI1 that down-regulates Notch/Wnt signaling and inhibits tumor xenograft growth in vivo. Using a fluorescence polarization (FP) competition assay, we identified gossypolone (Gn) with a > 20-fold increase in Ki value compared to (-)-gossypol. We validated Gn binding to MSI1 using surface plasmon resonance, nuclear magnetic resonance, and cellular thermal shift assay, and tested the effects of Gn on colon cancer cells and colon cancer DLD-1 xenografts in nude mice. RESULTS: In colon cancer cells, Gn reduced Notch/Wnt signaling and induced apoptosis. Compared to (-)-gossypol, the same concentration of Gn is less active in all the cell assays tested. To increase Gn bioavailability, we used PEGylated liposomes in our in vivo studies. Gn-lip via tail vein injection inhibited the growth of human colon cancer DLD-1 xenografts in nude mice, as compared to the untreated control (P < 0.01, n = 10). CONCLUSION: Our data suggest that PEGylation improved the bioavailability of Gn as well as achieved tumor-targeted delivery and controlled release of Gn, which enhanced its overall biocompatibility and drug efficacy in vivo. This provides proof of concept for the development of Gn-lip as a molecular therapy for colon cancer with MSI1/MSI2 overexpression.
Subject(s)
Colonic Neoplasms/drug therapy , Gossypol/analogs & derivatives , Nerve Tissue Proteins/antagonists & inhibitors , RNA-Binding Proteins/antagonists & inhibitors , Animals , Apoptosis/drug effects , Biological Products/administration & dosage , Cell Line, Tumor , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Gene Expression Regulation, Neoplastic/drug effects , Gossypol/administration & dosage , Humans , Liposomes/administration & dosage , Mice , Nerve Tissue Proteins/genetics , RNA-Binding Proteins/genetics , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Glioblastoma multiforme (GBM) is a poorly curable disease due to its heterogeneity that enables single cells to survive treatment regimen and initiate tumor regrowth. Although some progress in therapy has been achieved in the last years, the efficient treatment of GBMs is still a clinical challenge. Besides the standard therapeutic drug temozolomide (TMZ), quinoline-based antimalarial drugs such as hydroxychloroquine (HCQ) and BH3 mimetics such as AT101 were considered as possible drugs for GBM therapy. METHODS: We investigated the effects of sequentially applied single and combined TMZ, HCQ and AT101 treatments in a long-term stimulation GBM in vitro model. We performed all investigations in parallel in human astrocytes and two differentially TMZ-responsive human GBM cell lines and adjusted used drug concentrations to known liquor/plasma concentrations in patients. We determined amounts of dead cells and still remaining growth rates and depicted our results in a heatmap-like summary to visualize which sequential long-term treatment schedule seemed to be most promising. RESULTS: We showed that sequential stimulations yielded higher cytotoxicity and better tumor growth control in comparison to single TMZ treatment. This was especially the case for the sequences TMZ/HCQ and TMZ + AT101/AT101 which was as effective as the non-sequential combination TMZ + AT101. Importantly, those affected both less and more TMZ-responsive glioma cell lines, whilst being less harmful for astrocytes in comparison to single TMZ treatment. CONCLUSIONS: Sequential treatment with mechanistically different acting drugs might be an option to reduce side effects in long-term treatment, for example in local administration approaches.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Glioblastoma/drug therapy , Cell Growth Processes/drug effects , Cell Line, Tumor , Dacarbazine/administration & dosage , Dacarbazine/analogs & derivatives , Drug Administration Schedule , Drug Synergism , Glioblastoma/pathology , Gossypol/administration & dosage , Gossypol/analogs & derivatives , Humans , Hydroxychloroquine/administration & dosage , TemozolomideABSTRACT
Misregulation of BCL-2 family of proteins renders a survival signal to withstand cytotoxic anticancer drugs and is often found in drug resistant cells. The drug resistance phenotype is also associated with an enhancement of cancer stem cell-like (CSC) characteristics. Thus, inhibition of anti-apoptotic BCL-2 family proteins has been proposed as a possible antineoplastic strategy, and BCL-2 inhibitors are currently being clinically trailed in patients with leukemia, lymphoma or non-small cell lung cancer. However, the effects of BCL-2 inhibitors on drug resistant breast cancer have not yet been elucidated. In the present study, the effect of sabutoclax, a pan-active BCL-2 protein family antagonist, on two chemoresistant breast cancer cell lines was assessed. We found that sabutoclax showed a significant cytotoxic activity on chemoresistant breast cancer cells both in vitro and in vivo. When chemotherapeutic agents were combined with sabutoclax, strong synergistic antiproliferative effects were observed. Sabutoclax induced the blockage of BCL-2, MCL-1, BCL-xL and BFL-1, which in turn led to caspase-3/7 and caspase-9 activation and modulation of Bax, Bim, PUMA and survivin expression. Furthermore, sabutoclax effectively eliminated the CSC subpopulation and reduced sphere formation of drug-resistant cells through down-regulation of the IL-6/STAT3 signaling pathway. A similar effect was observed in a small panel of nine breast tumors ex vivo. Our findings indicate that sabutoclax partially overcomes the drug resistance phenotype of breast cancer cells by reactivation of apoptosis, mediated by the inhibition of several anti-apoptotic BCL-2 family proteins, and eliminates CSCs by abolition of the IL-6/STAT3 pathway. This offers a strong rationale to explore the therapeutic strategy of using sabutoclax alone or in combination for chemotherapy-nonresponsive breast cancer patients.
Subject(s)
Breast Neoplasms/drug therapy , Doxorubicin/administration & dosage , Drug Resistance, Neoplasm/drug effects , Gossypol/analogs & derivatives , Neoplastic Stem Cells/drug effects , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Synergism , Female , Gene Expression Regulation, Neoplastic/drug effects , Gossypol/administration & dosage , Gossypol/pharmacology , Humans , Interleukin-6/metabolism , MCF-7 Cells , Mice , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The aim of the present study was to analyze seminal quality of young bulls subjected to different frequencies of gossypol supplementation. Forty-eight Nellore bulls, with 19 months of age and weighing 357.8⯱â¯7.2â¯kg, were used in this study. Animals were fed with 10.5â¯kg of standard supplement containing free-gossypol from whole cottonseed (WCS) at the following frequency: 3x/week (G3x), 5x/week (G5x) or 7x/week (G7x - Control). Additionally, a negative control was provided, and the treated animals received only mineral supplement (MM) ad libtum. The experiment lasted for 84 days and semen was collected at the beginning and at the end for analysis and cryopreservation. Fresh semen was used for initial analysis and plasma membrane integrity and sperm morphology were also determined. General motility using computer assisted sperm analysis (CASA), plasma and acrosomal membranes integrity, mitochondrial activity, and induced oxidative stress were assessed in post-thawed semen. The study design was completely randomized. Parametric data were analyzed by ANOVA and non-parametric data by the Wilcoxon test, using the statistical program SAS. Level of significance was set at 5%. Supplementation with WCS, regardless the frequency, increased total (Pâ¯=â¯.009) and head (Pâ¯=â¯.005) defects in comparison to animals receiving only forage and mineral supplement. Infrequent supplementation, particularly 5 times in the week (G5X), increased head (Pâ¯=â¯.026) and midpiece (Pâ¯=â¯.014) abnormalities. Sperm motility in fresh semen was lower in animals that received daily supplementation than those supplemented on alternate days (Pâ¯=â¯.021). Additionally, animals supplemented daily showed lower percentage of spermatozoa with intact acrosome compared to those supplemented on alternate days (Pâ¯=â¯.005). Thus, regardless the frequency of supplementation, free-gossypol supplementation affects sperm quality. Although the amount of free gossypol supplied weekly was the same among treatments, daily supplementation compromised sperm kinetics, differently from infrequent supplementation that led to sperm defects developed during spermatogenesis.
Subject(s)
Animal Feed , Cattle , Gossypol/administration & dosage , Gossypol/toxicity , Reproduction/drug effects , Animal Feed/toxicity , Animal Nutritional Physiological Phenomena , Animals , Cryopreservation/methods , Cryopreservation/veterinary , Dietary Supplements , Dose-Response Relationship, Drug , Drug Administration Schedule , Male , Semen/cytology , Semen/drug effects , Semen Analysis/veterinary , Semen Preservation/veterinary , Sexual Maturation/drug effects , Spermatogenesis/drug effectsABSTRACT
Gossypol was considered a promising male contraceptive but finally failed due to two side effects: hypokalemia and the irreversibility of its contraceptive effect. Here we demonstrate that sustained zero-order release could be a solution for these problems. The in vitro release of gossypol from gossypol/PEG layer-by-layer films follows a perfect zero-order kinetics. In vivo tests indicate that the films can maintain the plasma drug concentration constant in male SD rats for â¼20 days for a 30-bilayer film. The plasma drug concentration is 2 orders of magnitude lower than the peak plasma drug concentration when administered orally and the daily dose is >50-fold lower than the commonly used contraceptive oral dose. However, significant antifertility effects were still observed. Furthermore, hypokalemia was not observed, and the antifertility effects can be reversed after a recovery period. The results suggest that zero-order release can significantly improve the desired antifertility effect of gossypol and, meanwhile, significantly reduce its side effects. We envision the drug could be developed to be an effective, safe, and reversible male contraceptive by zero-order release.
Subject(s)
Contraceptive Agents, Male/adverse effects , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Gossypol/adverse effects , Gossypol/pharmacokinetics , Animals , Contraceptive Agents, Male/administration & dosage , Contraceptive Agents, Male/blood , Contraceptive Agents, Male/pharmacokinetics , Drug Carriers/administration & dosage , Drug Implants/administration & dosage , Drug Implants/chemistry , Drug Implants/pharmacokinetics , Gossypol/administration & dosage , Gossypol/blood , Hypokalemia/chemically induced , Male , Rats, Sprague-DawleyABSTRACT
Exposure of the lung to ionizing radiation that occurs in radiotherapy, as well as after accidental or intentional mass casualty incident can result in pulmonary fibrosis, which has few treatment options. Pulmonary fibrosis is characterized by an accumulation of extracellular matrix proteins that create scar tissue. Although the mechanisms leading to radiation-induced pulmonary fibrosis remain poorly understood, one frequent observation is the activation of the profibrotic cytokine transforming growth factor-beta (TGF-ß). Our laboratory has shown that the metabolite lactate activates latent TGF-ß by a reduction in extracellular pH. We recently demonstrated that lactate dehydrogenase-A (LDHA), the enzyme that produces lactate, is upregulated in patients with radiation-induced pulmonary fibrosis. Furthermore, genetic silencing of LDHA or pharmacologic inhibition using the LDHA inhibitor gossypol prevented radiation-induced extracellular matrix secretion in vitro through inhibition of TGF-ß activation. In the current study, we hypothesized that LDHA inhibition in vivo prevents radiation-induced pulmonary fibrosis. To test this hypothesis, C57BL/6 mice received 5 Gy total-body irradiation plus 10 Gy thoracic irradiation from a 137Cs source to induce pulmonary fibrosis. Starting at 4 weeks postirradiation, mice were treated with 5 mg/kg of the LDHA inhibitor gossypol or vehicle daily until sacrifice at 26 weeks postirradiation. Exposure to radiation resulted in pulmonary fibrosis, characterized by an increase in collagen content, fibrosis area, extracellular matrix gene expression and TGF-ß activation. Irradiated mice treated with gossypol had significantly reduced fibrosis outcomes, including reduced collagen content in the lungs, reduced expression of active TGF-ß, LDHA and the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α). These findings suggest that inhibition of LDHA protects against radiation-induced pulmonary fibrosis, and may be a novel therapeutic strategy for radiation-induced pulmonary fibrosis.
Subject(s)
Gossypol/administration & dosage , L-Lactate Dehydrogenase/antagonists & inhibitors , Pulmonary Fibrosis/immunology , Pulmonary Fibrosis/prevention & control , Radiation Pneumonitis/immunology , Radiation Pneumonitis/prevention & control , Animals , Cytokines/immunology , Dose-Response Relationship, Drug , Male , Mice , Mice, Inbred C57BL , Pulmonary Fibrosis/pathology , Radiation Dosage , Radiation Pneumonitis/pathology , Radiation Protection/methods , Radiation Tolerance/drug effects , Radiation-Protective Agents/administration & dosage , Treatment OutcomeABSTRACT
PURPOSE: The aim of tumor-specific chemoradiotherapy is to achieve synergistic anticancer effects with clinically acceptable toxicity. Our previous studies showed that Pluronic P85 augments radiation cancer cell killing of (±)-gossypol in vitro. In this study, the radiosensitizing effect of (-)-gossypol, more potent Bcl protein inhibitor, with Pluronic P85 was investigated. MATERIALS AND METHODS: The inhibitory effect of (-)-gossypol solubilized Pluronic P85 with 0-8 Gy of radiation on clonogenic survival rate of A549 human lung adenocarcinoma cells was investigated in vitro. The anticancer effect of (-)-gossypol-solubilized Pluronic P85 with fractionated radiation of 15 Gy was assessed by A549 tumor-bearing mice. RESULTS: (-)-Gossypol-loaded Pluronic P85 was found to be a more potent radiosensitizer in vitro. Pluronic P85 increased the anti-proliferative activity of (-)-gossypol against A549 cells (82 ± 42 versus 190 ± 60 nM). In addition, the combination of P85 and (-)-gossypol effectively reduced clonogenic survival of A549 cells: (11 ± 5%) compared to (-)-gossypol and P85 alone (62 ± 27% and 93 ± 13%, respectively), and enhanced radiation cancer cell killing. In vivo, P85 (200 mg/kg/day) and (-)-gossypol (15 mg/kg/day) could be safely injected intravenously over 5 days and enhanced radiation-related tumor control in an A549 xenograft model. CONCLUSION: Pluronic P85 and (-)-gossypol act as a novel dual agent radiosensitizer and holds promise as a chemoradiotherapeutic strategy.
Subject(s)
Chemoradiotherapy/methods , Gossypol/administration & dosage , Neoplasms, Experimental/pathology , Neoplasms, Experimental/therapy , Poloxalene/administration & dosage , Radiation-Sensitizing Agents/administration & dosage , A549 Cells , Animals , Antineoplastic Agents/administration & dosage , Cell Survival/drug effects , Cell Survival/radiation effects , Dose-Response Relationship, Drug , Female , Mice , Mice, Nude , Treatment OutcomeABSTRACT
Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin.
Subject(s)
Adenosine Triphosphate/metabolism , Aldehyde Dehydrogenase/antagonists & inhibitors , Carcinoma, Non-Small-Cell Lung/drug therapy , Gossypol/administration & dosage , Lung Neoplasms/drug therapy , Phenformin/administration & dosage , Action Potentials , Aldehyde Dehydrogenase/metabolism , Animals , Aspartic Acid/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cytosol/metabolism , Female , Humans , Lung Neoplasms/metabolism , Malates/metabolism , Membrane Potential, Mitochondrial , Mice , Mice, Inbred BALB C , Mice, Nude , Mitochondria/metabolism , NAD/metabolism , NADP/metabolism , Neoplasm Transplantation , Oxidative Phosphorylation , Oxidoreductases Acting on CH-NH Group Donors , RNA, Small Interfering/metabolismABSTRACT
BACKGROUND: AT-101 is a BCL-2 Homolog domain 3 mimetic previously demonstrated to have tumoricidal effects in advanced solid organ malignancies. Given the evidence of activity in xenograft models, treatment with AT-101 in combination with docetaxel is a therapeutic doublet of interest in metastatic head and neck squamous cell carcinoma. PATIENTS AND METHODS: Patients included in this trial had unresectable, recurrent, or distantly metastatic head and neck squamous cell carcinoma (R/M HNSCC) not amenable to curative radiation or surgery. This was an open label randomized, phase II trial in which patients were administered AT-101 in addition to docetaxel. The three treatment arms were docetaxel, docetaxel plus pulse dose AT-101, and docetaxel plus metronomic dose AT-101. The primary endpoint of this trial was overall response rate. RESULTS: Thirty-five patients were registered and 32 were evaluable for treatment response. Doublet therapy with AT-101 and docetaxel was well tolerated with only 2 patients discontinuing therapy due to treatment related toxicities. The overall response rate was 11 % (4 partial responses) with a clinical benefit rate of 74 %. Median progression free survival was 4.3 months (range: 0.7-13.7) and overall survival was 5.5 months (range: 0.4-24). No significant differences were noted between dosing strategies. CONCLUSION: Although met with a favorable toxicity profile, the addition of AT-101 to docetaxel in R/M HNSCC does not appear to demonstrate evidence of efficacy.
Subject(s)
Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Gossypol/analogs & derivatives , Head and Neck Neoplasms/drug therapy , Taxoids/therapeutic use , Adult , Aged , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Disease-Free Survival , Docetaxel , Drug Administration Schedule , Female , Gossypol/administration & dosage , Gossypol/adverse effects , Gossypol/therapeutic use , Humans , Male , Middle Aged , Neoplasm Recurrence, Local/drug therapy , Taxoids/administration & dosage , Taxoids/adverse effects , Treatment OutcomeABSTRACT
Prostate cancer (PCa) is the most common type of cancer among males. Although survival rate of early-stage PCa is high, treatment options are very limited for recurrent disease. In this study, the possible synergistic cytotoxic and apoptotic effect of octreotide in combination with AT-101 was investigated in DU-145 hormone and drug refractory prostate cancer cell line. To enlighten the action mechanisms of the combination treatment, expression levels of somatostatin receptors 2 and 5 (SSTR2 and SSTR5) were also investigated. Cell viability was measured by XTT assay. Apoptosis was assessed through DNA fragmentation analysis and caspase 3/7 assay. mRNA and protein levels of SSTR2 and SSTR5 were evaluated by qRT-PCR and western blot analysis, respectively. Octreotide in combination with AT-101 inhibited cell viability and induced apoptosis synergistically in DU-145 cells as compared to any agent alone. Combination treatment increased both SSTR2 and SSTR5 mRNA and protein levels in DU-145 cells. The data suggest that this combination therapy may be a good candidate for patients with advanced metastatic PCa do not respond to androgen deprivation.