Clinical trials of R-(-)-gossypol (AT-101) in newly diagnosed and recurrent glioblastoma: NABTT 0602 and NABTT 0702.

PURPOSE: AT-101 is an oral bcl-2 family protein inhibitor (Bcl-2, Bcl-XL, Mcl-1, Bcl-W) and potent inducer of proapoptotic proteins. A prior study of the parent compound, racemic gossypol, demonstrated objective and durable responses in patients with malignant glioma. AT-101 has demonstrated synergy with radiation in animal models. The objectives of trial NABTT 0602 were to determine the MTD of AT-101 concurrent with temozolomide (TMZ) and radiation therapy (RT) (Arm I) and to determine the MTD of AT-101 when given with adjuvant TMZ after completion of standard chemoradiation (Arm 2). Separately in trial NABTT 0702, the survival and response rates of single agent AT-101 were evaluated in patients with recurrent glioblastoma. METHODS: In NABTT 0602 Phase I, a 3+3 design was used to define MTDs after maximal safe resection, patients with newly diagnosed glioblastoma received standard concurrent RT (60 Gy) and TMZ 75 mg/m2/day followed by adjuvant TMZ 150-200 mg/m2 days 1-5 in 28-day cycles (Stupp regimen). In Arm I, AT-101 was administered M-F during the six weeks of RT beginning 20 mg qd. In Arm 2, concurrent with each adjuvant cycle of TMZ, AT-101 was administered at a starting dose of 20 mg, days 1-21 followed by 7-day break for a maximum of 6 cycles. The PK blood samples were collected in the first three patients in each cohort of arm 1. In NABTT 0702 patients with recurrent glioblastoma received 20 mg p.o. per day for 21 of 28 days in repeated cycles to assess overall survival (OS). RESULTS: A total of sixteen patients were enrolled on the two study arms of NABTT 0602. In Arm 1 AT-101 was escalated from 20 to 30 mg where one of six patients experienced DLT (grade 3 GI ulcer). On Arm 2 one patient treated at 20 mg experienced DLT (grade 3 ileus, nausea and diarrhea). The cohort was expanded to include seven patients without observation of DLT. PK results were consistent with drug levels from non-CNS studies. At study closure six patients are still alive. The median survival times for Arm I and Arm II are 15.2 months and 18.2 months, respectively. In NABTT 0702 fifty-six patients were enrolled and forty-three were eligible for imaging response. Sixteen patients (29%) had stable disease as best response and one partial response was observed. The median OS with single agent AT-101 was 5.7 months (95%CI: 3.8-7.6 months) for patients with rGBM. CONCLUSIONS: AT-101 can be safely administered with radiation therapy and TMZ in patients with newly diagnosed glioblastoma without toxicity unique to patients with CNS tumors. Because of toxicity observed in non-CNS AT-101 clinical trials, further dose-escalation was not attempted. The recommended dose for future studies that utilize continual AT-101 exposure is 20 mg days M-F concurrent with RT/TMZ and 20 mg days 1-21 for each 28-day cycle of TMZ. AT-101 has limited activity as a single agent in unselected patients with recurrent glioblastoma. Future trials should attempt to better understand resistance mechanisms and consider combination therapy.

Effect of feeding increasing levels of whole cottonseed on milk and milk components, milk fatty acid profile, and total-tract digestibility in lactating dairy cows.

Dietary fat is fed to increase energy intake and provide fatty acids (FA) to support milk fat production. Oilseeds contain unsaturated FA that increase the risk for biohydrogenation-induced milk fat depression, but FA in whole cottonseed (WCS) are expected to be slowly released in the rumen and thus have a lower risk for biohydrogenation-induced milk fat depression. Our hypothesis was that increasing dietary WCS would increase milk fat yield by providing additional dietary FA without induction of milk fat depression. Four primiparous and 8 multiparous lactating Holstein cows, 136 ± 35 and 127 ± 4 DIM, respectively, were arranged in a replicated 4 × 4 Latin square design with 21-d periods. Treatments were WCS provided at 0%, 3.4%, 6.8%, and 9.9% of dietary dry matter, and WCS was substituted for cottonseed hulls and soybean meal to maintain dietary fiber and protein. Treatment did not change milk yield. There was a treatment-by-parity interaction for milk fat percent and yield with a quadratic decreased in primiparous cows but no effect of WCS in multiparous cows. Cottonseed linearly increased milk fat trans-10 18:1 in primiparous cows but not in multiparous cows. Increasing WCS increased milk preformed (18C) FA yield and partially overcame the trans-10 18:1 inhibition of de novo FA synthesis in the primiparous cows. Apparent transfer of 18C FA from feed to milk decreased in all cows as WCS increased, but the magnitude of the change was greater in primiparous cows. Increasing WCS decreased total-tract apparent dry matter, organic matter, and neutral detergent fiber digestibility. There was no change in total FA digestibility. However, 18C FA digestibility tended to be decreased in both parities and 16C FA digestibility was quadratically increased in multiparous cows but not changed in primiparous cows. Total fecal flow of intact WCS increased as WCS level increased, but fecal flow of intact seeds as a percentage consumed was similar across treatments. Fecal flow of intact seeds was greater in multiparous cows (4.3% vs. 1.1% of consumed). Plasma concentrations of glucose, nonesterified FA, triglycerides, and insulin were not changed. However, plasma urea-N increased with increasing WCS. Plasma gossypol increased with WCS (0.08-1.15 µg/mL) but was well below expected toxic levels. In conclusion, WCS maintained milk and milk component yield when fed at up to 9.9% of the diet to multiparous cows without concerns of gossypol toxicity, but primiparous cows were more susceptible to biohydrogenation-induced milk fat depression in the current trial. This highlights the interactions of parity with diet composition when feeding rumen-available unsaturated fat to dairy cows.

GhMYC1374 regulates the cotton defense response to cotton aphids by mediating the production of flavonoids and free gossypol.

Myelocytomatosis (MYC) transcription factors (TFs) in plants are well-known regulators of plant defense against herbivores. However, the role and mechanism of MYC TFs in cotton (Gossypium hirsutum L.) defense against cotton aphids (Aphis gossypii Glover) remain still elusive. Herein, on the basis of aphid-induced cotton transcriptome analysis, GhMYC1374, a cotton MYC2-like TF that was highly induced by cotton aphid attack, has been identified that confers cotton aphid resistance in cotton. GhMYC1374 was an intranuclear transcription factor with three domains: bHLH-MYC_N, RBR and bHLH_AtAIB_like. GhMYC1374 was induced under cotton aphid feeding, exogenous methyl jasmonate (MeJA) and salicylic acid (SA) treatments. GhMYC1374 transient overexpression in cotton plants enhanced cotton aphid-resistance, while GhMYC1374 silence through VIGS (virus induced gene silencing) decreased cotton aphid-resistance. GhMYC1374 transient overexpression of in cotton plants activated the phenylpropane pathway and promoted the synthesis of flavonoids, and resistance to thus enhanced the cotton resistance against aphids. In contrast, GhMYC1374 silence inhibited the biosynthesis of flavonoids. In addition, GhMYC1374 also positively activated the expression of the biosynthetic genes of free gossypol, leading to the high content of free gossypol. Taken together, our results suggest that GhMYC1374 is involved in the cotton defense response against cotton aphids by regulating the biosynthesis of flavonoids and free gossypol.

Inhibition of human glutathione transferase by catechin and gossypol: comparative structural analysis by kinetic properties, molecular docking and their efficacy on the viability of human MCF-7 cells.

Glutathione transferase Pi (GSTP1) expression is increased in many cancer types and is associated with multidrug resistance and apoptosis inhibition. Inhibitors of GSTP1-1 have the potential to overcome drug resistance and improve chemotherapy efficacy as adjuvant agents. This study investigated the effects of catechin and gossypol on human glutathione transferase Pi (GSTP1-1) activity and their cytotoxic effects on breast cancer cells (MCF-7) individually and in combination with tamoxifen (TAM). Gossypol effectively inhibited the enzyme with an IC50 value of 40 µM, compared to 200 µM for catechin. Gossypol showed stronger inhibition of GSTP1-1 activity (Ki = 63.3 ± 17.5 µM) compared to catechin (Ki = 220 ± 44 µM). Molecular docking analysis revealed their binding conformations to GSTP1-1, with gossypol binding at the subunit interface in an un-competitive manner and catechin showing mixed non-competitive inhibition. Gossypol had severe cytotoxic effects on both MCF-7 cells and normal BJ1 cells, while catechin had a weak cytotoxic effect on MCF-7 cells only. Combination therapy with TAM resulted in cytotoxicity of 27.3% and 35.2% when combined with catechin and gossypol, respectively. Gossypol showed higher toxicity to MCF-7 cells, but its strong effects on normal cells raised concerns about selectivity and potential side effects.

Gossypol synergises antiproliferative effect of sorafenib in metastatic lung cancer cells following Chou-Talalay algorithm.

Combination therapy has been proposed as a promising approach for lung cancer treatment, as it can enhance anticancer efficacy, and reduce dosages and adverse effects. This study aimed to explore the therapeutic potential of gossypol, a natural polyphenolic compound with sorafenib for treating lung cancer cells and elucidating its mechanism of action. The MTT assay was utilized to determine the IC50 of sorafenib and gossypol against A549 and NCI H460 cell lines. The Chou-Talaly algorithm was employed to determine the combination index (CI). A sub-effective concentration of sorafenib and gossypol was chosen to investigate the possibility of cytotoxic synergy. Autophagy biomarkers were identified using Western blotting, and the function of autophagy was determined using ATG5 siRNA. Results show that IC50 of sorafenib significantly reduced in A549 and NCI H460 cells when co-treated with gossypol. The combination treatment showed a synergistic cytotoxic effect against tested cell lines. The Chou-Talaly algorithm confirmed sorafenib's dose reduction index (DRI) up to 3.86. In A549 cells, combination treatment down-regulated p62 and up-regulated LC3-II, indicating the initiation of autophagy-dependent cytotoxicity. This was further confirmed by siRNA ATG5 knockdown. Additionally, the combination treatment exclusively targeted G0/G1 phase cancer cells. In conclusion, the combination of gossypol and sorafenib shows a synergistic increase in the cytotoxic effect by promoting autophagy and apoptosis.

Gossypol improves myocardial dysfunction caused by sepsis by regulating histone acetylation.

Gossypol is a polyphenol from the cotton plant with anti-inflammatory and anti-oxidation activities and can also function as a histone deacetylase (HDAC) inhibitor. Sepsis is an inflammatory disease with high mortality. Inflammation, oxidative stress, and epigenetic factors are involved in sepsis and its complications. The biological activities of gossypol strongly suggest the potential effects of gossypol on sepsis. In the present study, the beneficial effects of gossypol on sepsis were evaluated. We established a cecal ligation and puncture (CLP) mouse model of sepsis and treated CLP mice with gossypol. The survival rate, serum level of myocardial injury markers, and myocardial level of oxidation markers were measured. We also administered gossypol to lipopolysaccharide (LPS)-treated primary cardiomyocytes. The production of pro-inflammatory cytokines, activation of protein kinase B (AKT) and IκB kinase (IKK), acetylation of histone, and expression of HDACs were measured. Gossypol prevented the death of CLP mice and ameliorated myocardial damage in CLP mice. Moreover, gossypol decreased oxidative factors, while promoting antioxidant production in CLP mice. Gossypol prevented LPS and cytosine-phosphate-guanosine-induced expression of pro-inflammatory cytokines, suppressed LPS-induced activation of AKT and IKK, inhibited histone acetylation, and decreased the expression of HDACs. In conclusion, gossypol ameliorates myocardial dysfunction in mice with sepsis.

Plant Polyphenol Gossypol Induced Cell Death and Its Association with Gene Expression in Mouse Macrophages.

Gossypol is a complex plant polyphenol reported to be cytotoxic and anti-inflammatory, but little is known about its effect on gene expression in macrophages. The objective of this study was to explore gossypol's toxicity and its effect on gene expression involved in the inflammatory response, glucose transport and insulin signaling pathways in mouse macrophages. Mouse RAW264.7 macrophages were treated with multiple concentrations of gossypol for 2-24 h. Gossypol toxicity was estimated by MTT assay and soluble protein content. qPCR analyzed the expression of anti-inflammatory tristetraprolin family (TTP/ZFP36), proinflammatory cytokine, glucose transporter (GLUT) and insulin signaling genes. Cell viability was greatly reduced by gossypol, accompanied with a dramatic reduction in soluble protein content in the cells. Gossypol treatment resulted in an increase in TTP mRNA level by 6-20-fold and increased ZFP36L1, ZFP36L2 and ZFP36L3 mRNA levels by 26-69-fold. Gossypol increased proinflammatory cytokine TNF, COX2, GM-CSF, INFγ and IL12b mRNA levels up to 39-458-fold. Gossypol treatment upregulated mRNA levels of GLUT1, GLUT3 and GLUT4 genes as well as INSR, AKT1, PIK3R1 and LEPR, but not APP genes. This study demonstrated that gossypol induced macrophage death and reduced soluble protein content, which was accompanied with the massive stimulation of anti-inflammatory TTP family and proinflammatory cytokine gene expression, as well as the elevation of gene expression involved in glucose transport and the insulin signaling pathway in mouse macrophages.

Cobalt Ferrite-Gossypol Coordination Nanoagents with High Photothermal Conversion Efficiency Sensitizing Chemotherapy against Bcl-2 to Induce Tumor Apoptosis.

Gossypol is a chemotherapeutic drug that can inhibit the anti-apoptotic protein Bcl-2, but the existing gossypol-related nanocarriers cannot well solve the problem of chemotherapy resistance. Based on the observation that gossypol becomes black upon Fe3+ coordination, it is hypothesized that encasing gossypol in glyceryl monooleate (GMO) and making it coordinate cobalt ferrite will not only improve its photothermal conversion efficiency (PCE) but also help it enter tumor cells. As the drug loading content and drug encapsulation efficiency of gossypol are 10.67% (w/w) and 96.20%, the PCE of cobalt ferrite rises from 14.71% to 36.00%. The synergistic therapeutic effect finally induces tumor apoptosis with a tumor inhibition rate of 96.56%, which is 2.99 and 1.47 times higher than chemotherapy or photothermal therapy (PTT) alone. PTT generated by the GMO nanocarriers under the irradiation of 808 nm laser can weaken tumor hypoxia, thereby assisting gossypol to inhibit Bcl-2. In addition, the efficacy of nanocarriers is also evaluated through T2 -weighted magnetic resonance imaging. Observations of gossypol-induced apoptosis in tissue slices provide definitive proof of chemotherapy sensitization, indicating that such coordination nanocarriers can be used as an effective preclinical agent to enhance chemotherapy.

Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells.

Glioblastoma (GBM) is a poorly treatable disease due to the fast development of tumor recurrences and high resistance to chemo- and radiotherapy. To overcome the highly adaptive behavior of GBMs, especially multimodal therapeutic approaches also including natural adjuvants have been investigated. However, despite increased efficiency, some GBM cells are still able to survive these advanced treatment regimens. Given this, the present study evaluates representative chemoresistance mechanisms of surviving human GBM primary cells in a complex in vitro co-culture model upon sequential application of temozolomide (TMZ) combined with AT101, the R(-) enantiomer of the naturally occurring cottonseed-derived gossypol. Treatment with TMZ+AT101/AT101, although highly efficient, yielded a predominance of phosphatidylserine-positive GBM cells over time. Analysis of the intracellular effects revealed phosphorylation of AKT, mTOR, and GSK3ß, resulting in the induction of various pro-tumorigenic genes in surviving GBM cells. A Torin2-mediated mTOR inhibition combined with TMZ+AT101/AT101 partly counteracted the observed TMZ+AT101/AT101-associated effects. Interestingly, treatment with TMZ+AT101/AT101 concomitantly changed the amount and composition of extracellular vesicles released from surviving GBM cells. Taken together, our analyses revealed that even when chemotherapeutic agents with different effector mechanisms are combined, a variety of chemoresistance mechanisms of surviving GBM cells must be taken into account.

Involvement of necroptosis in the selective toxicity of the natural compound (±) gossypol on squamous skin cancer cells in vitro.

Cutaneous basal and squamous cell carcinoma reflect the first and second most common type of non-melanoma skin cancer, respectively. Especially cutaneous squamous cell carcinoma has the tendency to metastasize, finally resulting in a rather poor prognosis. Therapeutic options comprise surgery, radiation therapy, and a systemic or targeted chemotherapy. There are some good treatment results, but overall, the response rate of newly developed drugs is still modest. Drug repurposing represents an alternative approach where already available and clinically approved substances are used, which originally intended for other clinical benefits. In this context, we tested the effect of the naturally occurring polyphenolic aldehyde (±) gossypol with concentrations between 1 and 5 µM on the invasive squamous cell carcinoma cell line SCL-1 and normal human epidermal keratinocytes. Gossypol treatment up to 96 h resulted in a selective cytotoxicity of SCL-1 cells (IC50: 1.7 µM, 96 h) compared with normal keratinocytes (IC50: ≥ 5.4 µM, 96 h) which is mediated by mitochondrial dysfunction and finally leading to necroptotic cell death. Taken together, gossypol shows a high potential as an alternative anticancer drug for the treatment of cutaneous squamous cell carcinoma.

A biopolymeric mesh enriched with PLGA microparticles loaded with AT101 for localized glioblastoma treatment.

Current treatment strategies for glioblastoma (GBM) including surgical resection and adjuvant radio/chemotherapy result in a limited progression-free survival time of patients due to rapidly occurring tumor recurrences. The urgent need for more effective treatments has led to the development of different approaches for localized drug delivery systems (DDSs) offering the advantages of reduced systemic side effects. A promising candidate for the treatment of GBMs is AT101, the R-(-)-enantiomer of gossypol due to its ability to induce apoptosis or trigger autophagic cell death in tumor cells. Here, we present an alginate-based drug-releasing mesh ladened with AT101-loaded PLGA microspheres (AT101-GlioMesh). The AT101-loaded PLGA microspheres were fabricated using an oil-in-water emulsion solvent evaporation method obtaining a high encapsulation efficiency. The drug-loaded microspheres enabled the release of AT101 over several days at the tumor site. The cytotoxic effect of the AT101-loaded mesh was evaluated using two different GBM cell lines. Strikingly, encapsulation of AT101 in PLGA-microparticles and subsequent embedding in GlioMesh resulted in a sustained delivery and more efficient cytotoxic effect of AT101 on both GBM cell lines. Thus, such a DDS holds promise for GBM therapy likely by preventing the development of tumor recurrences.

Effects of cottonseed meal on performance, gossypol residue, liver function, lipid metabolism, and cecal microbiota in geese.

A total of 240 28-d-old male goslings were used to investigate the effects of cottonseed meal (CSM) on performance, gossypol residue, liver function, lipid metabolism, and cecal microbiota. All birds were randomly allotted into five groups (eight goslings/replicate, six replicates/group) and subjected to a 35-d experiment. Five isonitrogenous and isoenergetic diets were formulated to produce diets in which 0% (control), 25% (CSM25), 50% (CSM50), 75% (CSM75), and 100% (CSM100) of protein from soybean meal was replaced by protein from CSM. The free gossypol contents in the five diets were 0, 44, 92, 135, and 183 mg/kg, respectively. Dietary CSM did not affect the growth performance from 29 to 63 d and carcass traits at 63 d (P > 0.05). Liver gossypol residues were influenced (P < 0.05) by dietary CSM and increased linearly (P < 0.05) and quadratically (P < 0.05) as dietary CSM increased. The malondialdehyde content of the liver was lower in the CSM100 group than in the other groups (P < 0.05). Serum triglyceride and low-density lipoprotein cholesterol were influenced (P < 0.05) by dietary CSM and increased linearly (P < 0.05) with increasing dietary CSM. Dietary CSM altered (P < 0.05) the composition of some fatty acids in the liver and breast muscle. The concentration of linolenic acid and Σn-3 polyunsaturated fatty acid (PUFA) in the liver and breast muscle decreased linearly, but the Σn-6/Σn-3 PUFA ratio increased linearly with increasing dietary CSM (P < 0.05). Dietary CSM affected (P < 0.05) the hepatic gene expression of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and apolipoprotein B (ApoB). As the dietary CSM concentration increased, the hepatic gene expression of FAS increased linearly (P < 0.05) and quadratically (P < 0.05), but the hepatic gene expression of ACC and ApoB increased linearly (P < 0.05). The CSM diet decreased the relative abundance of the Bacteroidota and Bacteroides (P < 0.05), and the CSM50 diet increased the relative abundance of the Firmicutes and Colidextribacter (P < 0.05) compared to the control group. Overall, these results show that dietary CSM has no adverse effects on the performance of goslings from 29 to 63 d. However, CSM affected organismal lipid metabolism, reduced products' edible value, and adaptively altered cecum microbiota.

The shortage of feed resources and the rising price have become one of the significant challenges for animal husbandry worldwide. Considering the strong tolerance and adaptability to roughage of geese, less expensive crop byproducts are used in goose feed by animal nutritionists. Cottonseed meal (CSM) is a potential substitute for soybean meal, and the main concern for its use in poultry feed is free gossypol. This study aimed to investigate the effects of CSM on the performance, gossypol residue, liver function, lipid metabolism, and cecal microbiota in geese. Results showed that dietary CSM has no adverse effects on the performance and liver function of goslings. However, gossypol residue in goose liver increased with increasing dietary CSM. Besides, CSM affected organismal lipid metabolism, altered the tissue fatty acid composition, and adaptively changed cecum microbial microbiota. In summary, CSM is a good dietary protein source for geese, but further attention may be needed to its use for the edible value of goose products.

(-)- Gossypol Inhibition of Musashi-Mediated Forgetting Improves Memory and Age-Dependent Memory Decline in Caenorhabditis elegans.

Musashi RNA-binding proteins (MSIs) retain a pivotal role in stem cell maintenance, tumorigenesis, and nervous system development. Recently, we showed in C. elegans that Musashi (MSI-1) actively promotes forgetting upon associative learning via a 3'UTR-dependent translational expression of the Arp2/3 actin branching complex. Here, we investigated the evolutionary conserved role of MSI proteins and the effect of their pharmacological inhibition on memory. Expression of human Musashi 1 (MSI1) and Musashi 2 (MSI2) under the endogenous Musashi promoter fully rescued the phenotype of msi-1(lf) worms. Furthermore, pharmacological inhibition of human MSI1 and MSI2 activity using (-)- gossypol resulted in improved memory retention, without causing locomotor, chemotactic, or learning deficits. No drug effect was observed in msi-1(lf) treated worms. Using Western blotting and confocal microscopy, we found no changes in MSI-1 protein abundance following (-)- gossypol treatment, suggesting that Musashi gene expression remains unaltered and that the compound exerts its inhibitory effect post-translationally. Additionally, (-)- gossypol suppressed the previously seen rescue of the msi-1(lf) phenotype in worms expressing human MSI1 specifically in the AVA neuron, indicating that (-)- gossypol can regulate the Musashi pathway in a memory-related neuronal circuit in worms. Finally, treating aged worms with (-)- gossypol reversed physiological age-dependent memory decline. Taken together, our findings indicate that pharmacological inhibition of Musashi might represent a promising approach for memory modulation.

Knockdown of cytochrome P450 gene CYP6AB12 based on nanomaterial technology reduces the detoxification ability of Spodoptera litura to gossypol.

In insects, cytochrome P450 monooxygenases (P450s or CYPs) play an important role in the detoxification and metabolism of exogenous plant allelochemicals. In this study, a P450 gene CYP6AB12 was identified and characterized from Spodoptera litura. The cDNA contains an open reading frame (ORF) encoding 511 amino acid residues. CYP6AB12 was expressed at different ages of S. litura, with the highest levels found in the third and fourth instar larvae. Its highest expression was found in the midgut and fat body of fourth instar larvae fed with gossypol. Moreover, these expression levels were substantially increased compared with those from larvae fed with control diet. Gene silencing was then conducted by smearing dsRNA mixed with nanomaterials onto the cuticle. CYP6AB12 expression was significantly decreased in the midgut and fat body, and the net weight increase was substantially lower than that of the control group, indicating that the treatment group had more sensitivity to gossypol than the control. These results reveal that CYP6AB12 plays an important role in the detoxification and metabolism of gossypol, thus further confirming that P450s have a broad ability to detoxify and metabolize plant allelochemicals. It provides an important molecular basis for the exploration of detoxification metabolism and pest control of S. litura.

Identification of Bcl2 as a Stably Expressed qPCR Reference Gene for Human Colon Cancer Cells Treated with Cottonseed-Derived Gossypol and Bioactive Extracts and Bacteria-Derived Lipopolysaccharides.

Cottonseed contains many bioactive molecules including plant polyphenols. Cottonseed value might be increased by providing high-value bioactive polyphenols for improving nutrition and health. However, there was a lack of molecular evidence for cottonseed bioactivity in mammalian cells. One widely used method for evaluating the bioactivity of natural products is quantitative real-time-PCR (qPCR). The selection of stably expressed internal reference genes is a crucial task of qPCR assay for data analysis. The rationale for reference gene selection is that a lower standard deviation of the cycle of threshold (Cq) among the treatments indicates a more stable expression of the gene. The objective of this study was to select reference genes in human colon cancer cells (COLO 205) treated with cottonseed-derived gossypol and bioactive extracts along with bacterial endotoxin lipopolysaccharides (LPS). SYBR Green qPCR was used to analyze the mRNA levels of a wide range of biomarkers involved in glucose transport, lipid biosynthesis, inflammatory response, and cancer development. qPCR data (10,560 Cq values) were generated from 55 genes analyzed from 64 treatments with triplicate per treatment for each gene. The data showed that B-cell lymphoma 2 (Bcl2) mRNA was the most stable among the 55 mRNAs analyzed in the human colon cancer cells. Glyceraldehyde 3 phosphate dehydrogenase (Gapdh) and ribosome protein L32 (Rpl32) mRNAs were not good qPCR references for the colon cancer cells. These observations were consistent regardless of the treatment comparison between gossypol and LPS, glanded and glandless seed extracts, seed coat and kernel extracts, or treatment for 8 and 24 h. These results suggest that Bcl2 is a preferable reference gene for qPCR assays in human colon cancer cells treated with cottonseed-derived gossypol and bioactive extracts as well as LPS. The extensive qPCR results firmly support the conclusion that the Bcl2 gene is stably expressed at the mRNA level in the human colon cancer cells regardless of the treatment, suggesting that Bcl2 gene expression is not regulated at the mRNA level but at the post-transcriptional level. These results should facilitate studies designated to evaluate bioactivity on gene expression regulation by cottonseed molecules and other natural and synthetic molecules for nutrition and health uses.

Gossypol Broadly Inhibits Coronaviruses by Targeting RNA-Dependent RNA Polymerases.

Outbreaks of coronaviruses (CoVs), especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have posed serious threats to humans and animals, which urgently calls for effective broad-spectrum antivirals. RNA-dependent RNA polymerase (RdRp) plays an essential role in viral RNA synthesis and is an ideal pan-coronaviral therapeutic target. Herein, based on cryo-electron microscopy and biochemical approaches, gossypol (GOS) is identified from 881 natural products to directly block SARS-CoV-2 RdRp, thus inhibiting SARS-CoV-2 replication in both cellular and mouse infection models. GOS also acts as a potent inhibitor against the SARS-CoV-2 variant of concern (VOC) and exerts same inhibitory effects toward mutated RdRps of VOCs as the RdRp of the original SARS-CoV-2. Moreover, that the RdRp inhibitor GOS has broad-spectrum anti-coronavirus activity against alphacoronaviruses (porcine epidemic diarrhea virus and swine acute diarrhea syndrome coronavirus), betacoronaviruses (SARS-CoV-2), gammacoronaviruses (avian infectious bronchitis virus), and deltacoronaviruses (porcine deltacoronavirus) is showed. The findings demonstrate that GOS may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and other coronavirus outbreaks.

Effects of cottonseed meal on growth performance, liver redox status, and serum biochemical parameters in goslings at 1 to 28 days of age.

BACKGROUND: Cottonseed meal (CSM), a relatively rich source of protein and amino acids, is used as an inexpensive alternative to soybean meal (SBM) in poultry diets. However, the toxicity of free gossypol in CSM has been a primary concern. The present study was conducted to investigate the effects of CSM on growth performance, serum biochemical parameters, and liver redox status in goslings at 1 to 28 days of age. Three hundred 1-day-old male goslings were randomly divided into 5 groups (10 goslings/pen, 6 replicate pens/group) and subjected to a 28-day experiment. Five isonitrogenous and isoenergetic diets were formulated such that 0% (control), 25% (CSM25), 50% (CSM50), 75% (CSM75), and 100% (CSM100) of protein from SBM was replaced by protein from CSM. The free gossypol contents in the five diets were 0, 56, 109, 166, and 222 mg/kg, respectively. RESULTS: The results showed that dietary CSM was associated with linear decreases in body weight, average daily feed intake and average daily gain and linear increases in the feed-to-gain ratio from 1 to 28 days of age (P < 0.001). As the dietary CSM concentration increased, a numerical increase was found in the mortality of goslings. According to a single-slope broken-line model, the breakpoints for the average daily gain of dietary free gossypol concentration on days 1 to 14, 15 to 28, and 1 to 28 occurred at 23.63, 14.78, and 18.53 mg/kg, respectively. As the dietary CSM concentration increased, serum albumin (P < 0.001) concentrations decreased linearly and serum uric acid (P = 0.011) increased linearly. The hydroxyl radical scavenging ability (P = 0.002) and catalase (P < 0.001) and glutathione peroxidase (P = 0.001) activities of the liver decreased linearly with increasing dietary CSM. However, dietary CSM did not affect the concentrations of reactive oxygen metabolites, malondialdehyde, or protein carbonyl in the liver. CONCLUSIONS: The increasing dietary CSM increased the concentration of free gossypol and altered the composition of some amino acids in the diet. A high concentration of CSM reduced the growth performance of goslings aged 1 to 28 days by decreasing feed intake, liver metabolism, and antioxidant capacity. From the primary concern of free gossypol in CSM, the tolerance of goslings to free gossypol from CSM is low, and the toxicity of free gossypol has a cumulative effect over time.

Identification of the gossypol derivatives as androgen receptor inhibitor.

Prostate cancer (PCa) is the most frequently diagnosed male malignant tumor and remains the second leading cause of male cancer mortality in western countries. The development of novel antiandrogens to circumvent the drug resistance in anti-PCa treatment is highly demanded. Herein, we identified that gossypol (GOS) specificly inhibited the AR signaling. Further optimization of GOS derivatives led to the discovery of compound XY-32. XY-32 efficiently inhibits AR signaling with the IC50 of 1.23 µM. XY-32 downregulates both the full-length AR and the AR variable splice AR-V7 via suppressing the mRNA expression. It inhibits the proliferation of CRPC cells such as the LNCaP cells, the PC-3 cells, and enzalutamide resistance 22Rv1 cells. The work demonstrates the GOS derivatives represent a novel series of anti-androgen to conquer the acquired AR mutations or AR splice variants induced drug resistance of mCRPC.

Anticancer effects of putative and validated BH3-mimetic drugs in head and neck squamous cell carcinomas: An overview of current knowledge.

The purpose of this review was to summarise available literature concerning the anticancer effects of both putative and validated BH3-mimetics in head and neck squamous cell carcinomas. A literature search was performed and studies assessing malignant cell lines, xenograft models, and/or humans were considered eligible. A total of 501 studies were identified, of which 40 were included. One phase-II clinical trial assessing gossypol (combined with docetaxel) was found. The remaining 39 preclinical studies investigated cell lines and/or xenograft models involving the use of six validated BH3-mimetics (A-1210477, A-1331852, ABT-737, navitoclax, S63845, venetoclax) and six putative BH3-mimetics (ApoG2, gossypol, obatoclax, sabutoclax, TW-37, and YC137). In preclinical settings, most validated BH3-mimetics were capable of inducing apoptosis (in-vitro) and tumour growth inhibition (in-vivo). The majority of putative BH3-mimetics were also capable of inducing cell death, although important off-target effects, such as autophagy induction, were also described. Combinations with conventional anticancer drugs, ionising radiation, or multiple BH3-mimetics generally resulted in enhanced anticancer effects, such as increased sensitivity to apoptotic stimuli, especially considering some cell lines that showed resistance to either treatment alone. In conclusion, although clinical data are still insufficient to evaluate the anticancer effects of BH3-mimetics in head and neck squamous cell carcinomas, promising results in preclinical settings were observed concerning induction of cell death and inhibition of tumour growth. Therefore, further clinical trials are highly encouraged.

NMR-based metabolomics reveals tissue metabolic responses to tetramethoxy gossypol in cottonseed oil.

BACKGROUND: Cottonseed oil is one of the most widely consumed cooking oils because of its high nutritional benefits and relatively low price. The present study evaluated the effects of tetramethoxy gossypol (TMG), a rarely reported degradation product of free gossypol produced in crudely extracted cottonseed oil, on the metabolic responses of liver, heart, spleen, kidney and lung tissues in rats using proton nuclear magnetic resonance (1 H NMR) spectroscopy combined with chemometric and bioinformatics techniques. RESULTS: Endogenous low-molecular-weight metabolites in rat liver, heart, spleen, kidney and lung tissues were profiled by 1 H NMR spectroscopy. The unsupervised principal components analysis and the supervised orthogonal partial least squares discriminant analysis revealed that the metabolic profiles in liver samples were greatly changed after TMG administration. Twenty significantly changed liver metabolites were screened out and further evaluated by receiver operating characteristic curve analysis, which were closely related to amino acid, glutathione, energy and lipid metabolism. CONCLUSION: Concerning the potential chronic exposure to TMG in cottonseed oil and other cottonseed products, the cumulative effects of dietary TMG on tissues, especially the liver, should be noted when improving the quality control standard of cottonseed oil. © 2022 Society of Chemical Industry.