Coleus vettiveroides ethanolic root extract induces cytotoxicity by intrinsic apoptosis in HepG2 cells.

Hepatocellular carcinoma (HCC) contributes to more than 80% of all primary cancers globally and ranks fourth in cancer-related deaths, due to the lack of an effective, definite therapeutic drug. Coleus vettiveroides (CV) has been used in Indian traditional medicine to treat diabetes, liver ailments, skin diseases, leukoderma, and leprosy. This study investigates the anticancer effect of CV ethanolic root extract in HepG2 cells. HepG2 cells were treated with CV extract, and its cytotoxicity was analyzed by MTT assay. AO/EB staining, propidium iodide staining, DCFH-DA assay, phalloidine staining, flow cytometry, and qPCR studies were performed for ROS expression, apoptosis and cell cycle analysis. The phytochemical analysis confirmed the presence of quercetin and galangin in CV root extract. The results showed that CV inhibited the proliferation of HepG2 cells, with altered cellular and nuclear morphology. CV was also found to increase intracellular ROS levels and oxidative stress markers in HepG2 cells. CV significantly altered the actin microfilament distribution in HepG2 cells and caused cell cycle arrest at the sub G0 -G1 phase. CV also induced mitochondria-mediated apoptosis, as evidenced by increased expression of p53, Bax, cytochrome C, Apaf-1, PARP, caspase-3 and caspase-9, and downregulated Bcl-2 expression. Therefore, CV exerts its anticancer effect by inducing mitochondrial dysfunction, oxidative stress, cytoskeletal disorganization, cell cycle arrest, and mitochondria-mediated apoptosis, and it could be a potent therapeutic option for HCC.

Nelumbo nucifera Leaf Extract Induces Cytotoxicity in Osteosarcoma Saos-2 Cells.

Background Osteosarcoma is the eighth most common cancer and its prevalence in children makes it a global concern. Existing medications and treatments like high-dose methotrexate possess harmful side effects. Therefore, novel herbal drugs like Nelumbo nucifera are of utmost importance. Aim  To analyze a novel anticancer herbal drug, Nelumbo nucifera leaf extract for its cytotoxic potential against osteosarcoma.  Materials and method Nelumbo nucifera leaf extract was prepared. Saos-2 Cells (human osteosarcoma cell line) were treated with Nelumbo nucifera leaf extract (25, 50, 75, 100, 125, and 150 µg/ml) for 24 hours which were then subjected to MTT assay, morphological analysis and DAPI staining. Results The results suggested that Nelumbo nucifera leaf extract had a concentration-dependent cytotoxic effect on Saos-2 cell line. The extract significantly reduced the number of viable cells, inhibited proliferation and induced morphological changes in Saos-2 cells.  Conclusion Nelumbo nucifera has the potential to induce cytotoxicity against osteosarcoma cell lines and hence, this study provides a novel therapeutic regimen for the treatment of osteosarcoma.

Coleus vettiveroides ethanolic root extract protects against thioacetamide-induced acute liver injury in rats.

Acute liver injury is caused by various factors, including oxidative stress and inflammation. Coleus vettiveroides, an ayurvedic medicinal plant, is known to possess antioxidant, antibacterial, and antidiabetic properties. In this current study, we investigated the protective effect of C. vettiveroides ethanolic root extract (CVERE) against thioacetamide (TAA)-induced acute liver injury in rats. A single dose of TAA (300 mg/kg, b.w., i.p.) was administered to induce acute liver injury. The treatment groups of rats were concurrently treated with CVERE (125 and 250 mg/kg, b.w., p.o.) and silymarin (100 mg/kg, b.w., p.o.), respectively. After 24 h of the experimental period, TAA-induced liver injury was confirmed by increased activity of serum transaminases and malondialdehyde levels in liver tissue, decreased levels of antioxidants, upregulated expression of the inflammatory marker gene, and altered liver morphology. Whereas CVERE simultaneous treatment inhibited hepatic injury and prevented the elevation of serum aspartate and alanine transaminases, alkaline phosphatase, and lactate dehydrogenase activities. CVERE attenuated TAA-induced oxidative stress by suppressing lipid peroxidation and restoring antioxidants such as superoxide dismutase, catalase, and reduced glutathione. Further, CVERE treatment was found to inhibit nuclear factor κB-mediated inflammatory signaling, as indicated by downregulated pro-inflammatory cytokines including tumor necrosis factor-α and interleukin-1ß. Our findings suggest that CVERE prevents TAA-induced acute liver injury by targeting oxidative stress and inflammation.

Concurrent administration of farnesol protects acetaminophen-induced acute hepatic necrosis in mice.

Acetaminophen (APAP) is known to cause acute liver injury and acute liver failure in Western countries. This study investigates the protective role of farnesol (FAR) (C15 H26 O), a natural sesquiterpene alcohol in essential oils, against APAP-induced acute liver necrosis in mice. Mice were injected with a single dose of APAP (300 mg/kg) via an intraperitoneal route. Different groups of mice were concurrently treated with a single dose of FAR 25 mg/kg, FAR 50 mg/kg, and N-acetylcysteine. APAP administration caused a significant increase in transaminase activities and malondialdehyde (MDA) levels in the serum and liver tissue, respectively, with a concomitant decrease in intracellular antioxidants, including reduced glutathione (GSH) in the liver tissue. APAP intoxication upregulated proinflammatory cytokines such as tumor necrosis factor-α, interleukin-1ß (IL-1ß), IL-6, nuclear factor-κB (NF-κB), and IκB kinase ß in the liver tissue. FAR and N-acetylcysteine (NAC) administrations concurrently with APAP prevented serum transaminase increase in serum and MDA levels in the liver tissue. A high dose of FAR and NAC treatments significantly inhibited GSH and other antioxidant depletion. FAR and NAC treatments also downregulated the expression of proinflammatory markers. FAR treatments protects against APAP-induced acute liver injury and offers antioxidant and anti-inflammatory effects by inhibiting the NF-κB pathway involved in the transcription of genes responsible for inflammatory cytokine synthesis.

Molecular Targets of Nimbolide for Anti-Cancer Therapy: An Updated Review.

Cancer is a major cause of death worldwide with an increasing incidence rate and is considered a major public health problem. Distance metastasis to other tissues, high toxicity, and drug resistance of cancer cells to chemotherapy demand novel therapeutic approaches to treat cancer. Natural compounds from medicinal plants have been studied for therapeutic use in various malignancies. Nimbolide is an active principal compound from Azadirachta indica, which is an Asian traditional medicinal plant utilized historically as a remedy for a variety of diseases due to its antioxidant, anti-inflammatory, anti-cancer, and antimicrobial properties. It is a limonoid triterpene possessing potent anti-cancer effects in various types of cancers. It has been reported to induce multiple cytotoxic effects in tumor cells by modulating the cell proliferation, cell cycle, apoptosis, and metastasis by altering the various molecular signaling pathways. In the present review, we summarized all the in vitro and in vivo studies reporting the molecular targets of nimbolide for the therapeutic approaches in different types of cancer cells. We analyzed research publications up to September 2021 on the effect of nimbolide in various malignancies and the molecular mechanism of action. Nimbolide targets different signaling pathways including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin like growth factor (IGF), Wingless and INT-1 (Wnt)/ß-catenin, mitogen-activated protein kinases (MAPK)/c-Jun N-terminal kinases (JNK), phosphoinositide 3-kinase (PI3K)/AKT, tumor necrosis factor-α (TNF-α)/nuclear factor kappa B (NF-κß), and death receptor 5 (DR5) in several cancer cells. Nimbolide's widespread availability and absence of side effects, as well as understanding the molecular mechanism of nimbolide's action, will be useful to develop a therapeutic agent against cancer.

A Molecular Insight into the Role of Antioxidants in Nonalcoholic Fatty Liver Diseases.

Nonalcoholic fatty liver disease (NAFLD) defines fat accumulation in the liver, and it is commonly associated with metabolic syndromes like diabetes and obesity. Progressive NAFLD leads to nonalcoholic steatohepatitis (NASH) and ultimately causes cirrhosis and hepatocellular carcinoma, and NASH is currently a frequent cause of liver transplantation. Oxidative stress is often contributed to the progression of NAFLD, and hence, antioxidants such as silymarin, silybin, or silibinin, pentoxifylline, resveratrol, and vitamins A, C, and E are used in clinical trials against NAFLD. Silymarin induces the peroxisome proliferator-activated receptor α (PPARα), a fatty acid sensor, which promotes the transcription of genes that are required for the enzymes involved in lipid oxidation in hepatocytes. Silybin inhibits sterol regulatory element-binding protein 1 and carbohydrate response element-binding protein to downregulate the expression of genes responsible for de novo lipogenesis by activating AMP-activated protein kinase phosphorylation. Pentoxifylline inhibits TNF-α expression and endoplasmic reticulum stress-mediated inflammatory nuclear factor kappa B (NF-κB) activation. Thus, it prevents NAFLD to NASH progression. Resveratrol inhibits methylation at Nrf-2 promoters and NF-κB activity via SIRT1 activation in NAFLD conditions. However, clinically, resveratrol has not shown promising beneficial effects. Vitamin C is beneficial in NAFLD patients. Vitamin E is not effectively regressing hepatic fibrosis. Hence, its combination with antifibrotic agents is used as an adjuvant to produce a synergistic antifibrotic effect. However, to date, none of these antioxidants have been used as a definite therapeutic agent in NAFLD patients. Further, these antioxidants should be studied in NAFLD patients with larger populations and multiple endpoints in the future.

Lagerstroemia speciosa (L.) Pers., ethanolic leaves extract attenuates dapsone-induced liver inflammation in rats.

Drug-induced liver injury is a common cause of acute liver failure. Dapsone is increasingly used in combination with rifampicin for the treatment of leprosy and also for several dermatological disorders. Clinically, abnormal liver function and focal bile duct destruction were reported after dapsone therapy. Lagerstroemia speciosa Pers., commonly known as Banaba has been traditionally used to treat various ailments including diabetes and obesity due to its antioxidant and anti-inflammatory efficacies. This study investigated the hepatoprotective effect of ethanolic banaba leaves extract (EBLE) against dapsone-induced hepatotoxicity in rats. Dapsone (30 mg/kg, i.p.) was administered twice daily for 30 days. In separate groups, rats were post-treated orally with EBLE (250 and 500 mg/kg) and silymarin (100 mg/kg) once daily for 30 days after dapsone administration. The marker enzymes of hepatotoxicity, oxidative stress markers, inflammatory markers and histopathology of liver were done. HPTLC analysis confirmed the presence of 12.87 µg of corosolic acid per mg of EBLE. Dapsone administration-induced significant (p < 0.001) elevation of marker enzymes of hepatotoxicity in serum. This treatment also increased lipid peroxidation (p < 0.001) and pro-inflammatory markers (tumor necrosis factor-alpha, transforming growth factor-beta, and nuclear factor kappa-B) expressions (p < 0.001) and decreased antioxidants (p < 0.001) such superoxide dismutase, catalase and glutathione in the liver tissue. All these abnormalities were significantly (p < 0.001) mitigated after EBLE (500 mg/kg) and silymarin post-treatments. The results of this study suggest that silymarin and EBLE can be used for dapsone-induced hepatotoxicity.

Sodium Valproate, a Histone Deacetylase Inhibitor, Provokes Reactive Oxygen Species-Mediated Cytotoxicity in Human Hepatocellular Carcinoma Cells.

BACKGROUND AND AIM: Sodium valproate (SV), a novel class of histone deacetylases (HDACs) inhibitors commonly used as an antiepileptic drug. HDAC inhibitors are known to possess anticancer potentials. In this study, we investigated the cytotoxic potential of SV in human hepatocellular carcinoma (HepG2 cells) cell line. METHODS: MTT assay was used to analyze cytotoxicity. Intracellular ROS and cytochrome c expression were analyzed by fluorescence microscopy. Morphology-related apoptosis was analyzed by dual staining with acridine orange/ethidium bromide. Caspase 3 protein expression was investigated by Western blotting analysis. RESULTS: Sodium valproate treatments in HepG2 cells caused significant and dose-dependent cytotoxicity. Intracellular ROS was remarkably increased in the cells which are treated with SV and caused early and late apoptosis as evidenced by dual staining. SV-treated cells expressed cytochrome c and caspase 3 protein expression. CONCLUSION: These results suggest the cytotoxic potentials of SV in HepG2 cells. This study may give an important clue for the inclusion of SV as an adjuvant along with standard anticancer agents after necessary in vivo and clinical studies.

Reactive Oxygen Species-Mediated Mitochondrial Dysfunction Triggers Sodium Valproate-Induced Cytotoxicity in Human Colorectal Adenocarcinoma Cells.

BACKGROUND: Colorectal cancer (CRC) is one of the frequently diagnosed cancers worldwide. Currently used chemotherapeutic drugs have several side effects. Histone deacetylase (HDAC) enzyme inhibitors possess potential anti-cancer effects. Therefore, we investigated the cytotoxic potential of sodium valproate, a HDAC inhibitor in human colorectal adenocarcinoma (HT-29) cells. METHODS: MTT assay was used to analyze the cytotoxicity of HT-29 cells. Intracellular reactive oxygen species (ROS) induction was evaluated by dichloro-dihydro-fluorescein diacetate staining. Dual staining with acridine orange/ethidium bromide was used to investigate the morphology-related apoptotic cell death. Mitochondrial membrane potential was analyzed by rhodamine 123 staining. E-cadherin protein expression was examined by immunofluorescence staining. RESULTS: Sodium valproate at 2 and 4 mM/mL treatments significantly induced cytotoxicity. Increased intracellular ROS expression was observed in the cells treated with sodium valproate. This treatment also induced mitochondrial dissipation, apoptosis-related morphological damage, and E-cadherin expression in HT-29 cells. CONCLUSIONS: Our present results suggest that sodium valproate is cytotoxic to HT-29 cells due to its pro-oxidative and apoptosis inducing potential. Sodium valproate can be used as an adjuvant along with standard chemotherapeutic agents in CRC patients after necessary in vivo and clinical studies.

Aqueous extract of Tamarindus indica fruit pulp exhibits antihyperglycaemic activity.

OBJECTIVE: Tamarindus indica Linn. (T.indica) is a well-known plant used in traditional medicine. The plant is popular for its antidiabetic activity. However, effect so f its aqueous fruit pulp extract on carbohydrate hydrolyzing enzymes and its glucose uptake potential were not explored. MATERIALS AND METHODS: The antidiabetic activity was assessed by in-vitro α-amylase and α-glucosidase inhibitory assays after preliminary phytochemical analysis. MTT assay was carried out to find cytotoxicity. Glucose uptake activity of the extract was carried out using L6 myotubes. RESULTS: The results showed a strong α-amylase inhibitory activity for the fruit pulp extract of T.indica compared to standard acarbose; the IC50 of the fruit pulp extract of T.indica and acarbose was 34.19 µg/ml 34.83µM. The extract also showed moderate α-glucosidase inhibitory activity. IC50 of the fruit pulp extract of T.indica and acarbose were 56.91µg/ml and 45.69µM respectively. The cytotoxicity assay showed IC50 of >300µg/ml and ≥1000µM for the fruit pulp extract of T.indica and metformin. The extract showed 63.99±0.08% glucose uptake in L6 myotubes whereas metformin and insulin at 10µg/ml and 10µM exhibited an uptake of 76.99±0.3% and 84.48±0.45% glucose, respectively. CONCLUSION: The study revealed that the fruit pulp extract of T.indica Linn does not show any cytotoxic effect and has very good α-amylase and good α-glucosidase inhibitory activities. The glucose uptake potential proves its postprandial hypoglycemic effect. Hence, it may be considered an antidiabetic agent for control of postprandial hyperglycemia.

Syringic acid induces apoptosis in human oral squamous carcinoma cells through mitochondrial pathway.

BACKGROUND: Syringic acid (SA) has long been used as traditional medicine and is known to have antioxidant, hepatoprotective, neuroprotective and anticancer effects. Studies regarding the anticancer effect of SA against squamous carcinoma cell (SCC)-25, human oral SCC (OSCC) line has not been studied. AIM: This study was aimed to evaluate the cytotoxic potentials of SA in SCC-25 cells. MATERIALS AND METHODS: Cytotoxic effect of SA was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte trazolium bromide assay, using concentrations of 25 and 50 µM/mL for 24 h. At the end of the treatment period, apoptotic markers such as caspase 3 and 9, bcl-2, bax and cytochrome c were evaluated by semiquantitative reverse transcription-polymerase chain reaction. SA-induced morphological changes were investigated by acridine orange/ethidium bromide dual staining. RESULTS: SA inhibited the proliferation and induced cytotoxicity in SCC-25 cells in a concentration-dependent manner. SA treatment caused apoptosis-related morphological changes as evidenced by the dual staining and the modulation of apoptotic marker gene expressions. SA treatments modulated bcl-2/bax homeostasis and increased the expressions of cytochrome c and caspases 3 and 9. CONCLUSION: SA specifically induces cell death and inhibits the proliferation in OSCC cells through intrinsic/mitochondrial apoptosis pathway, suggesting that SA may be an effective agent for the treatment of human OSCC.

Syzygium cumini extract induced reactive oxygen species-mediated apoptosis in human oral squamous carcinoma cells.

BACKGROUND: Syzygium cumini (L.) Skeels (jambolan) is commonly used in Indian traditional medicine to treat a variety of diseases such as obesity, diabetes etc. The cytotoxic potential of S. cumini (SC) against oral cancer cell line remains elusive. Therefore, in this study, we evaluated the cytotoxic effect of S. cumini in human oral squamous cell carcinoma (OSCC) cell line (SCC-25 cells). MATERIAL AND METHODS: Oral squamous cell carcinoma cells are treated with different concentrations (10, 20, and 40 µg/mL) of S. cumuni for 24 hours and cytotoxicity was analyzed by MTT assay. The intracellular reactive oxygen species (ROS) was measured using the indicator dye, 2',7'-dichlorofluorescin diacetate staining. Apoptosis-related morphological changes were evaluated by dual acridine orange/ethidium bromide (AO/EB) fluorescent staining and phosphatidylserine externalization was measured by annexin V assays. The protein and gene expression of cadherin-1 was evaluated by western blotting and PCR analysis. RESULTS: Syzygium cumini treatments caused cytotoxicity of OSCC cell line and induced intracellular ROS accumulation. This treatment also caused apoptosis-related morphological changes and externalization of phosphatidylserine in OSCC cells. Further, S. cumini treatments increased protein and gene expression of cadherin-1. CONCLUSION: Syzygium cumini extract inhibits the proliferation of OSCC cells and induces apoptosis through ROS accumulation and therefore, it could be used for the prevention of OSCC.

Silibinin alleviates N-nitrosodimethylamine-induced glutathione dysregulation and hepatotoxicity in rats.

The present study was designed to evaluate the hepatoprotective and antioxidant potentials of silibinin (SBN) against N-nitrosodimethylamine (DMN)-induced toxic insults in the rat liver. The liver damage was induced in Wistar albino rats by repeated administration of DMN (10 mg·kg(-1) b.w., i.p.) on 3 consecutive days per week for 3 weeks. SBN (100 mg·kg(-1) b.w., p.o.) was given daily to the DMN treated rats for two weeks. The marker enzymes of liver toxicity and second-line enzymic and non-enzymic antioxidants were evaluated in serum and liver tissues before and after SBN treatment. Histopathology of the liver was evaluated by H & E staining. The DMN treatment produced a progressive increase in all the serum marker enzymes (AST, ALT, ALP, LDH, and γ-GT), peaking on Day 21. This treatment produced highly significant decreases in all the second-line antioxidant parameters (GSH, GST, GR, GPx, and vitamins C and E). The SBN treatment significantly reversed the DMN-induced damages, towards normalcy. Histopathological studies confirmed the development of liver toxicity in DMN-treated rats, which was reversed by SBN treatment in corroboration with the aforementioned biochemical results, indicating the hepatoprotective and antioxidant properties of SBN. In conclusion, the DMN-induced degenerative changes in the liver were alleviated by SBN treatment and this protective ability may be attributed to its antioxidant, free radical scavenging, and membrane stabilizing properties.