Decursinol Angelate Inhibits Glutamate Dehydrogenase 1 Activity and Induces Intrinsic Apoptosis in MDR-CRC Cells.

Colorectal cancer (CRC) was the second most commonly diagnosed cancer worldwide and the second most common cause of cancer-related deaths in Europe in 2020. After CRC patients' recovery, in many cases a patient's tumor returns and develops chemoresistance, which has remained a major challenge worldwide. We previously published our novel findings on the role of DA in inhibiting the activity of GDH1 using in silico and enzymatic assays. No studies have been conducted so far to explain the inhibitory role of DA against glutamate dehydrogenase in MDR-CRC cells. We developed a multidrug-resistant colorectal cancer cell line, HCT-116MDR, after treatment with cisplatin and 5-fluorouracil. We confirmed the MDR phenotype by evaluating the expression of MDR1, ABCB5, extracellular vesicles, polyploidy, DNA damage response markers and GDH1 in comparison with parental HCT-116WT (HCT-116 wild type). Following confirmation, we determined the IC50 and performed clonogenic assay for the efficacy of decursinol angelate (DA) against HCT-116MDR (HCT-116 multidrug resistant). Subsequently, we evaluated the novel interactions of DA with GDH1 and the expression of important markers regulating redox homeostasis and cell death. DA treatment markedly downregulated the expression of GDH1 at 50 and 75 µM after 36 h, which directly correlated with reduced expression of the Krebs cycle metabolites α-ketoglutarate and fumarate. We also observed a systematic dose-dependent downregulation of MDR1, ABCB5, TERT, ERCC1 and γH2AX. Similarly, the expression of important antioxidant markers was also downregulated. The markers for intrinsic apoptosis were notably upregulated in a dose-dependent manner. The results were further validated by flow cytometry and TUNEL assay. Additionally, GDH1 knockdown on both HCT-116WT and HCT-116MDR corresponded to a decreased expression of γH2AX, catalase, SOD1 and Gpx-1, and an eventual increase in apoptosis markers. In conclusion, inhibition of GDH1 increased ROS production, decreased cell proliferation and increased cell death.

CopA3 treatment suppressed multidrug resistivity in HCT-116 cell line by p53-induced degradation of hypoxia-inducible factor 1α.

The major reason for multidrug resistance is the failure of chemotherapy in many tumors, including colon cancer. Hypoxia-inducible factor (HIF)-1α is a crucial transcription factor that simulates multiple cellular response to hypoxia. HIF-1α has been known to play a vital role towards tumor resistance; however, its mechanism of action is still not fully elucidated. N this study, we found that HIF-1α remarkably modulated drug resistance-associated proteins upon CopA3 peptide treatment against colon cancer cells. Abnormal rates of tumor growth along with high metastatic potential lacks the susceptibility towards cellular signals is a key characteristic in many tumor types. Moreover, in growing tumors, cells are exposed to insufficient nutrient supply and low oxygen availability. These stress force them to switch into adaptable and aggressive phenotypes. Our study investigated the interaction of HIF-1α and MDR gene association upon CopA3 treatment in the tumor microenvironment. We demonstrate that the multidrug resistance gene is associated with tumor resistance to chemotherapeutics, which upon CopA3 treatment promotes p53 activation and proteasomal degradation of HIF-1α, effecting the angiogenesis response to hypoxia. p53 downregulation augments HIF-1-dependent transcriptional activation of VEGF in response to oxygen deprivation.

Therapeutic propensity of ginsenosides Rg1 and Rg3 in rhabdomyolysis-induced acute kidney injury and renohepatic crosstalk in rats.

BACKGROUND: Ginseng is a traditional herbal medicine used for thousands of years in Southeast Asian countries because of its medicinal properties. Ginsenosides Rg1 and Rg3 have demonstrated therapeutic properties against a broad spectrum of diseases. PURPOSE: Here in this study, we investigated the therapeutic efficacy of Rg1 and Rg3 in alleviating glycerol-induced acute kidney injury, also known as rhabdomyolysis-induced acute kidney injury (RAKI). METHODS: AKI was induced in male Wistar rats through intramuscular injection of 10 mL/kg glycerol and simultaneous oral treatment of ginsenosides Rg1 and Rg3 for 3 days. We also evaluated the therapeutic potential of Rg1 and Rg3 on human embryonic kidney epithelial (HEK-293). Cell viability and LDH assay were performed on HEK-293 cells to evaluate the toxicity of Rg1 and Rg3. Evaluation of important kidney damage markers such as creatinine and blood urea nitrogen (BUN) was carried out at different time points from the rat serum. Histopathological analysis was performed on kidney tissues. We also performed experiments such as ELISA assay, immunohistochemistry, immunofluorescence staining, COMET assay, western blotting, TUNEL assay, and flow cytometry to obtain results. RESULTS: Rg1 and Rg3 significantly downregulated the expression of kidney damage markers such as creatinine and BUN in a dose-dependent manner. Histopathological analysis revealed damage across the glomerulus, tubules, and collecting duct rendering the kidney dysfunctional in glycerol treatment groups. However, Rg1 and Rg3 treated groups showed a significant reduction in tubular necrosis at both 10 and 20 mg/kg. There was also a sharp downregulation of oxidative and ER stress markers. Additionally, we observed nuclear translocation of Nrf2 which were more prominent in kidney tissues. Rg1 and Rg3 were also able to mitigate apoptotic cell death in vitro and in vivo evaluated through immunofluorescence staining for p53, TUNEL assay, flow cytometry, and immunoblotting for intrinsic apoptosis markers. CONCLUSION: In summary, we conclude that Rg1 and Rg3 exhibited natural therapeutic remedy against AKI.

Evaluation of decursin and its isomer decursinol angelate as potential inhibitors of human glutamate dehydrogenase activity through in silico and enzymatic assay screening.

Glutaminolysis is a typical hallmark of malignant tumors across different cancers. Glutamate dehydrogenase (GDH, GLUD1) is one such enzyme involved in the conversion of glutamate to α-ketoglutarate. High levels of GDH are associated with numerous diseases and is also a prognostic marker for predicting metastasis in colorectal cancer. Therefore, inhibiting GDH can be a crucial therapeutic target. Here in this study, we performed molecular docking analysis of 8 different plants derived single compounds collected from pubChem database for screening and selected decursin (DN) and decursinol angelate (DA). We performed molecular dynamics simulation (MD), monitored the stability, interaction for protein and docked ligand at 50 ns, and evaluated the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculation on the twoselected compounds along with a standard inhibitor epigallocatechin gallate (EGCG) as reference. The final results showed the formation of stable hydrogen bond interactions by DN and DA in the residues of R400 and Y386 at the ADP activation site of GDH, which was important for the selective inhibition of GDH activity. Additionally, the total binding energy of DN and DA were -115.5 kJ/mol and -106.2 kJ/mol, which was higher than the standard reference GDH inhibitor EGCG (-92.8 kJ/mol). Furthermore, biochemical analysis for GDH inhibition substantiated our computational results and established DN and DA as novel GDH inhibitor. The percentage of IC50 inhibition for DN and DA were 1.035 µM and 1.432 µM. Conclusively, DN and DA can be a novel therapeutic drug for inhibition of glutamate dehydrogenase.

Rhabdomyolysis-induced acute kidney injury and concomitant apoptosis induction via ROS-mediated ER stress is efficaciously counteracted by epigallocatechin gallate.

Rhabdomyolysis induced acute kidney injury (RIAKI) is a life-threatening condition responsible for approximately 19-58% of AKI cases worldwide. We performed an intramuscular injection of glycerol (10 mL/kg) in male wistar rats to induce AKI. Epigallocatechin gallate (EGCG) was administered for 3 consecutive days to evaluate its protective effects. We observed significant downregulation in serum creatinine, blood urea nitrogen (BUN) and LDH at different time points on EGCG treatment groups in a dose-dependent manner. Similarly, H&E staining also revealed that EGCG was able to reduce the formation of damaged tubules and tubular necrosis which was prominently spread throughout the kidney tissue of glycerol treatment group. Concomitantly, we observed upregulated inflammation, ER stress and elevated oxidative stress in the glycerol treated group only, which was significantly normalized upon EGCG treatment in both in vitro and in vivo studies. The occurrence of apoptosis in kidney tubules was found to be relatively higher in glycerol treated group and H2O2 treated HEK-293 cells. The results obtained after EGCG treatment revealed a significant decrease in apoptotic cell population, which was further validated by immunofluorescence staining against p53 and comet assay in HEK-293 cells and p53 IHC in kidney tissues. Western blotting also revealed a systemic downregulation of intrinsic mitochondrial apoptotic pathway markers such as bax, bcl-2, pro and cleaved caspase 3, caspase 9 and PARP1. Additionally, the results for flow cytometry analysis and TUNEL assay corroborated apoptotic equilibrium. Conclusively, we reckon EGCG as a multi-therapeutic natural product that can be used the for treatment of AKI.

Moringa oleifera mitigates ethanol-induced oxidative stress, fatty degeneration and hepatic steatosis by promoting Nrf2 in mice.

BACKGROUND: Moringa oleifera (M. oleifera) is cultivated throughout the world and it is known by numerous regional names and is consumed as medication for various diseases such as hypertension, diabetes, HIV and is potential source of nutrients and natural antioxidants making it among the most useful trees. METHODS: We evaluated the therapeutic potential of M. oleifera on ethanol-induced fatty liver. The mice were treated with 30% ethanol (EtOH) alone or in combination with different concentration of M. oleifera extracts (100, 200 and 400 mg/kg). We performed biochemical estimation for the serum of important liver damage markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and triglyceride (TG). We performed histopathological analysis from the liver tissues of different mice groups. We also performed ELISA assay, western blotting analysis and SPECT imaging to obtain our results. RESULTS: The results for serum (AST, p < 0.0001), (ALT, p < 0.0006) and triglyceride (TG, p < 0.0003) were found to be significantly reduced in all doses of M. oleifera extract treatment groups in comparison with the ethanol group. H&E staining analysis and scoring revealed a significant reduction in lipid droplet accumulation and a significant reduction of liver steatosis (p < 0.0001), lobular inflammation (p < 0.0013), ballooning (p < 0.0004) and immunohistochemistry for TNF-α. M. oleifera also ameliorated ethanol-induced oxidative stress evaluated through MDA (p < 0.0001), H2DCFDA, JC-1 staining and a significant down-regulation of CYP2E1 enzyme (p < 0.0001) in the 200 and 400 mg/kg groups in comparison with EtOH groups. M. oleifera extract also boosted the antioxidant response evaluated through total GSH assay (p < 0.0001) and nuclear translocation of Nrf2. Furthermore, we performed SPECT imaging and evaluated the liver uptake value (LUV) to assess the extent of liver damage. LUV was observed to be lower in the ethanol group, whereas LUV was higher in control and M. olifera treated groups. CONCLUSION: In summary, from this experiment we conclude that M. oleifera extract has the potential to ameliorate ethanol-induced liver damage.

Correction: Chang et al. Decursinol Angelate Arrest Melanoma Cell Proliferation by Initiating Cell Death and Tumor Shrinkage via Induction of Apoptosis. Int. J. Mol. Sci. 2021, 22, 4096.

The author wishes to make the following correction to this paper [...].

Quercetin enhances vitamin D2 stability and mitigate the degradation influenced by elevated temperature and pH value.

Vitamin D2 (vit. D2) is a nutraceutical essentially needed for good health. However, it is susceptible to oxygen and high temperature. The use of natural products such as bioflavonoids possessing anti-degradative effect of vit. D2 degradation has not been described before. A combinational effect of vit. D2 with quercetin showed a positive effect and inhibited vit. D2 degradation when exposed to high temperature (50 ℃ and 75 ℃) at different time points. The results obtained revealed vit. D2 degradation was drastically increased with longer incubation under thermal treatment. However, quercetin and vit. D2 groups were able to significantly inhibit the degradation of vit. D2 and stabilize it, evaluated through the retention percentage. We also exposed vit. D2 at solutions with different pH values (1, 4, 5, 7, 10). Quercetin exerted vit. D2 anti-degradation at different pH values as well as under thermal pressure at different time points. Conclusively, quercetin can be an effective way to reduce temperature and pH induced degradation of vit. D2.

Ultraviolet B-irradiated mushroom supplementation increased the Ca++ uptake and ameliorated the LPS-induced inflammatory responses in zebrafish larvae.

The harmful effects of excessive ultraviolet (UV) exposure are well known. However, moderate exposure to UV radiation is beneficial and required for active vitamin D synthesis in our body. People living in the coldest regions on the earth are unable to expose their skin to the solar UV radiation and, therefore, additional supplementation of Vitamin D2 is recommended. Mushrooms are one such consumable macrofungi, which has high vitamin content and therefore used in various traditional medicines. Particularly, UVB-irradiated mushrooms are rich in active vitamin D content and that is why recommended to include in the daily diets for the patients suffering from the problems associated with bone mineralization. In the present study, we evaluated the cytotoxic effect of mushroom extract (UVB-ME) (Lentinus edodes) treatment against MG-63 cells, HepG2 cells, and CCD 841 CoN cells. Furthermore, we elucidated the potential of UVB-ME on Ca++ uptake in osteoblast-like MG-63 cells. Next, we validated the response of Ca++ uptake on the growth and development of zebrafish larvae. In addition, the anti-inflammatory and immunomodulatory potential of UVB-ME treatment against lipopolysaccharide-induced inflammatory response was also analyzed in vivo. Collectively, the study suggested that dietary supplementation of UVB-irradiated mushroom is beneficial for bone calcification and could modulate the host immune system.

Decursinol Angelate Arrest Melanoma Cell Proliferation by Initiating Cell Death and Tumor Shrinkage via Induction of Apoptosis.

Melanoma is known to aggressively metastasize and is one of the prominent causes of skin cancer mortality. This study was designed to assess the molecular mechanism of decursinol angelate (DA) against murine melanoma cell line (B16F10 cells). Treatment of DA resulted in growth inhibition and cell cycle arrest at G0/G1 (p < 0.001) phase, evaluated through immunoblotting. Moreover, autophagy-related proteins such as ATG-5 (p < 0.0001), ATG-7 (p < 0.0001), beclin-1 (p < 0.0001) and transition of LC3-I to LC3-II (p < 0.0001) were markedly decreased, indicating autophagosome inhibition. Additionally, DA treatment triggered apoptotic events which were corroborated by the occurrence of distorted nuclei, elevated reactive oxygen species (ROS) levels and reduction in the mitochondrial membrane potential. Subsequently, there was an increase in the expression of pro-apoptotic protein Bax in a dose-dependent manner, with the corresponding downregulation of Bcl-2 expression and cytochrome C expression following 24 h DA treatment in A375.SM and B16F10 cells. We substantiated our results for apoptotic occurrence through flow cytometry in B16F10 cells. Furthermore, we treated B16F10 cells with N-acetyl-L-cysteine (NAC). NAC treatment upregulated ATG-5 (p < 0.0001), beclin-1 (p < 0.0001) and LC3-I to LC3-II (p < 0.0001) conversion, which was inhibited in the DA treatment group. We also noticed a systematic upregulation of important markers for progression of G1 cell phase such as CDK-2 (p < 0.029), CDK-4 (p < 0.036), cyclin D1 (p < 0.0003) and cyclin E (p < 0.020) upon NAC treatment. In addition, we also observed a significant fold reduction (p < 0.05) in ROS fluorescent intensity and the expression of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase-9 (p > 0.010) and cleaved caspase-3 (p < 0.0001). NAC treatment was able to ameliorate DA-induced apoptosis and cell cycle arrest to support our finding. Our in vivo xenograft model also revealed similar findings, such as downregulation of CDK-2 (p < 0.0001) and CDK-4 (p < 0.0142) and upregulation of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase 3 (p < 0.0001) and cleaved caspase 9 (p < 0.0001). In summary, our study revealed that DA is an effective treatment against B16F10 melanoma cells and xenograft mice model.

5-O-Demethylnobiletin Alleviates CCl4-Induced Acute Liver Injury by Equilibrating ROS-Mediated Apoptosis and Autophagy Induction.

Polymethoxyflavanoids (PMFs) have exhibited a vast array of therapeutic biological properties. 5-O-Demethylnobiletin (5-DN) is one such PMF having anti-inflammatory activity, yet its role in hepatoprotection has not been studied before. Results from in vitro study revealed that 5-DN did not exert a high level of cytotoxicity on HepG2 cells at 40 µM, and it was able to rescue HepG2 cell death induced by carbon tetrachloride (CCl4). Subsequently, we investigated acute liver injury on BALB/c mice induced by CCl4 through the intraperitoneal injection of 1 mL/kg CCl4 and co-administration of 5-DN at (1 and 2 mg/kg) by oral gavage for 15 days. The results illustrated that treatment with 5-DN attenuated CCl4-induced elevated serum aminotransferase (AST)/alanine aminotransferase (ALT) ratio and significantly ameliorated severe hepatic damage such as inflammation and fibrosis evidenced through lesser aberrations in the liver histology of 5-DN dose groups. Additionally, 5-DN efficiently counteracted and equilibrated the production of ROS accelerated by CCl4 and dramatically downregulated the expression of CYP2E1 vitally involved in converting CCl4 to toxic free radicals and also enhanced the antioxidant enzymes. 5-DN treatment also inhibited cell proliferation and inflammatory pathway abnormally regulated by CCl4 treatment. Furthermore, the apoptotic response induced by CCl4 treatment was remarkably reduced by enhanced Bcl-2 expression and noticeable reduction in Bax, Bid, cleaved caspase 3, caspase 9, and apaf-1 expression. 5-DN treatment also induced the conversion of LC3 and promoted the autophagic flux. Conclusively, 5-DN exhibited hepatoprotective effects in vitro and in vivo and prevented liver fibrosis induced by CCl4.

The inflammation response and risk associated with aflatoxin B1 contamination was minimized by insect peptide CopA3 treatment and act towards the beneficial health outcomes.

This study focused on the possible chemo-preventive effects of insect peptide CopA3 on normal human colon cells against the inflammation induced by the toxic environmental pollutant aflatoxin B1 (AFB1). In the study, we used CCD 841 CoN normal human colon cells to investigate the cytotoxic effect induced by AFB1 and elucidated the negative impact of AFB1 exposure on the cell cycle progression. Further, we also carried out the in-vivo experiment, where male BALB/c mice were administrated with AFB1 to induce inflammation associated cancer like phenotype and the dietary effect of CopA3 was evaluated on the early stages of AFB1-induced hepatotoxicity and inflammation in colon tissues. At the initiation stage, CopA3 was given along with water, which significantly decreased the inflammation in the liver and colon of AFB1 exposed mice model. Mice that received CopA3 alone showed enhanced activity of several antioxidant enzymes. In the post treatment stage, the CopA3 dosage remarkably increased the Ki-67 protein expression, indicating the enhancement in cell proliferation event and increased the number of apoptotic cells in colonic crypts, suggesting the capability of CopA3 treatment towards the epithelial cell turnover. Thus, CopA3 treatment shows its potential to inhibit the development of the early stages of AFB1-induced colon inflammation and hepatotoxicity in mice by inhibiting the DNA synthesis of the damaged and inflammatory cell and induced apoptosis for the clearance of damaged cells. Collectively, the results of this study suggest that CopA3 treatment may play a protective role against the mycotoxin induced inflammation.

Phorbol 12-Myristate 13-Acetate Induced Toxicity Study and the Role of Tangeretin in Abrogating HIF-1α-NF-κB Crosstalk In Vitro and In Vivo.

Phorbol 12-myristate 13-acetate (PMA) is a potent tumor promoter and highly inflammatory in nature. Here, we investigated the toxic effects of PMA on different model system. PMA (10 µg) caused chromosomal aberrations on the Allium cepa root tip and induced mitotic dysfunction. Similarly, PMA caused embryonic and larval deformities and a plummeted survivability rate on zebrafish embryo in a dose-dependent manner. Persistently, PMA treatment on immortalized human keratinocyte human keratinocyte (HaCaT) cells caused massive inflammatory rush at 4 h and a drop in cell survivability at 24 h. Concomitantly, we replicated a cutaneous inflammation similar to human psoriasis induced by PMA. Herein, we used tangeretin (TAN), as an antagonist to counteract the inflammatory response. Results from an in vivo experiment indicated that TAN (10 and 30 mg/kg) significantly inhibited PMA stimulated epidermal hyperplasia and intra-epidermal neutrophilic abscesses. In addition, its treatment effectively neutralized PMA induced elevated reactive oxygen species (ROS) generation on in vitro and in vivo systems, promoting antioxidant response. The association of hypoxia-inducible factor 1-alpha (HIF-1α)-nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB) crosstalk triggered by PMA enhanced PKCα-ERK1/2-NF-κB pathway; its activation was also significantly counteracted after TAN treatment. Conclusively, we demonstrated TAN inhibited the nuclear translocation of HIF-1α and NF-κB p65. Collectively, TAN treatment ameliorated PMA incited malignant inflammatory response by remodeling the cutaneous microenvironment.

Synergistic therapy with tangeretin and 5-fluorouracil accelerates the ROS/JNK mediated apoptotic pathway in human colorectal cancer cell.

Synergistic therapy is emerging as a promising strategy for improving the chemotherapeutic efficacy of anticancer drugs. Addition of adjuvants with standard anticancer drugs has shown successful reduction of adverse side effects. The synthetic drug 5-Fluorouracil (5-FU) shows several side effects upon prolonged chemotherapy, thereby restricting its long-term clinical application. Several studies have reported anticancer potential and anti-inflammatory activity of tangeretin (TAN) towards mammalian cells. Therefore, we investigate whether the combination of TAN with 5-FU increases their anticancer potential against colorectal cancer. In this study, we examined the synergistic activity of TAN and 5-FU on the viability of several human cancer and normal cells. Several possible mechanistic pathways were screened, and found that co-exposure of TAN and 5-FU accelerates oxidative-stress and increases endogenous-ROS generation, which sequentially triggers the DNA damage response and activates the apoptotic pathway, by down-regulating autophagy and DNA repair system in HCT-116 cells. TAN and 5-FU co-treatment also remarkably reduces the mitochondrial membrane potential, and sequentially decreases ATPase activity. Collectively, results indicate that combination of TAN and 5-FU significantly accelerates apoptosis via JNK mediated pathway. To our knowledge gained from literature, this study is the first to describe synergistic activity of TAN and 5-FU against colorectal cancer cells.

Decursinol angelate ameliorates 12-O-tetradecanoyl phorbol-13-acetate (TPA) -induced NF-κB activation on mice ears by inhibiting exaggerated inflammatory cell infiltration, oxidative stress and pro-inflammatory cytokine production.

Decursinol angelate (DA) is a pyranocoumarin purified from the roots of Angelica gigas. Here, we synthesized DA and determined its anti-inflammatory potential on TPA-induced mice ear inflammation. First, we evaluated the non-toxic behaviour of DA on HaCaT cells. Additionally, we observed the free radical scavenging potential of DA at 60 µM to be 50%. This finding was further supported by nitric oxide assay, malondialdehyde assay, H2DCFDA staining and western blotting analysis of antioxidant enzymes. DA also suppressed the activation and polarization of macrophage phagocytic activity on RAW 264.7 cells. We further evaluated the expression of ICAM-1, MCP-1, MIP-2 and MIP-1ß on in-vivo model system. Consequently, DA significantly reduced the production of NF-κB and COX-2 induced proinflammatory cytokine levels on TPA induced ear edema. Inhibition of MAPK and transcriptional factor NF-κB was also validated by western blotting analysis of p-ERK, p-p38, IKKα, IKKγ, IκBα, NF-κB-p65. Immunohistochemistry and immunofluorescence staining of NFκB-p65, TNF-α and IL-1ß were also performed to support the findings. Conclusively, these results suggest that topical administration of DA significantly inhibited the expression of pro-inflammatory cytokines by blocking the canonical NF-κB and MAPK pathway. Therefore, we suggest DA as a potent therapeutic compound against skin inflammation related diseases.