In-vitro Cytotoxicity Investigations for Phytoconstituents of Saudi Medicinal Plants With Putative Ocular Effects.

BACKGROUND: Metastatic secondary ocular tumors spread from systemic malignancies, including breast cancer. This study aimed to evaluate the cytotoxicity of extracts from 5 medicinal plants native to Saudi Arabia. METHODS: For preliminary activity screening, cytotoxicity using the MTT assay and selectivity index determinations were made for medicinal plant extracts against various cancer cell-lines. The most promising extract was subjected to GC-MS analysis to determine the phytochemical composition. Clonogenic assays were performed using the most promising extract to confirm the initial results. Finally, western blot analysis was used to determine the modulation in expression of survivin and P27 suppressor genes in the human breast adenocarcinoma (MCF7) cell-line to understand the potential mechanistic properties of the active plant extract. RESULTS: The 5 plant extracts showed various cytotoxic activity levels using IC50. The most active extract was found to be the leaves of Capparis spinosa L. (BEP-07 extract) against the MCF7 breast cancer cell-line (IC50 = 3.61 ± 0.99 µg/ml) and selectivity index of 1.17 compared to the normal human fetal lung fibroblast (MRC5) cells. BEP-07 extract showed a dose dependent clonogenic effect against the MCF7 colonies which was comparable with the effect of doxorubicin. BEP-07 extract caused a significant decrease of survivin and increase in P27 expression compared to control GAPDH at its highest dose (14 µg/ml). The GC-MS chromatogram of Capparis spinosa L. (BEP-07 extract) revealed the existence of 145 compounds, belonging to the diverse classes of phytoconstituents. Fatty acids and their derivatives represent 15.4%, whilst octadecanoic acid, 2,3-dihydroxypropyl ester was the principal component (7.9%) detected. CONCLUSION: Leaves of Capparis spinosa L. (BEP-07 extract) exhibited a significant cytotoxic effect particularly against breast cancer cells. It exhibited this effect through survivin inhibition and via P27 upregulation. The detected phytoconstituents in the plant extract might be involved in tested cytotoxic activity, while further investigations are required to complete the drug candidate profile.

Improved Glycaemic Control and Nephroprotective Effects of Empagliflozin and Paricalcitol Co-Therapy in Mice with Type 2 Diabetes Mellitus.

Herein, we measured the antidiabetic and nephroprotective effects of the sodium-glucose cotransporter-2 inhibitor (empagliflozin; SGLT2i) and synthetic active vitamin D (paricalcitol; Pcal) mono- and co-therapy against diabetic nephropathy (DN). Fifty mice were assigned into negative (NC) and positive (PC) control, SGLT2i, Pcal, and SGLT2i+Pcal groups. Following establishment of DN, SGLT2i (5.1 mg/kg/day) and/or Pcal (0.5 µg/kg/day) were used in the designated groups (5 times/week/day). DN was affirmed in the PC group by hyperglycaemia, dyslipidaemia, polyuria, proteinuria, elevated urine protein/creatinine ratio, and abnormal renal biochemical parameters. Renal SREBP-1 lipogenic molecule, adipokines (leptin/resistin), pro-oxidant (MDA/H2O2), pro-inflammatory (IL1ß/IL6/TNF-α), tissue damage (iNOS/TGF-ß1/NGAL/KIM-1), and apoptosis (TUNEL/Caspase-3) markers also increased in the PC group. In contrast, renal lipolytic (PPARα/PPARγ), adiponectin, antioxidant (GSH/GPx1/SOD1/CAT), and anti-inflammatory (IL10) molecules decreased in the PC group. Both monotherapies increased insulin levels and mitigated hyperglycaemia, dyslipidaemia, renal and urine biochemical profiles alongside renal lipid regulatory molecules, inflammation, and oxidative stress. While SGLT2i monotherapy showed superior effects to Pcal, their combination demonstrated enhanced remedial actions related to metabolic control alongside renal oxidative stress, inflammation, and apoptosis. In conclusion, SGLT2i was better than Pcal monotherapy against DN, and their combination revealed better nephroprotection, plausibly by enhanced glycaemic control with boosted renal antioxidative and anti-inflammatory mechanisms.

Antioxidative and Anti-Inflammatory Protective Effects of Fucoxanthin against Paracetamol-Induced Hepatotoxicity in Rats.

Paracetamol or acetaminophen (PAC) is a commonly used analgesic and antipyretic drug. It has been shown that overdoses beyond the therapeutic range can cause hepatotoxicity and acute liver injury. The most common cause of drug-induced liver injury (DILI) in Saudi Arabia and worldwide is paracetamol overdose. Fucoxanthin (FUC) is an allenic carotenoid that is found in edible brown seaweeds, and it has antioxidant and anti-inflammatory effects. Several studies have shown the potential therapeutic effects of FUC in diabetes, cancers, and inflammatory disorders. This study aims to investigate the protective effect of FUC against PAC-induced acute liver injury in rats. FUC was administered (100, 200, and 500 mg/kg, p.o.) for 7 days, and then the liver injury was induced by the administration of PAC (2000 mg/kg, oral). Blood and liver tissue samples were collected from PAC-positive untreated, treated, and negative control rats. Biochemical and inflammatory parameters in the blood were measured. In addition, RT-PCR, Western blotting, and immunohistochemistry were performed for liver tissue. The serum levels of liver biomarkers (ALT, AST, and ALP) increased after PAC-induced liver toxicity; FUC-treated rats showed lower levels compared to the positive control. There was an increase in the expression of TNF-α, IL-1, IL-6, NF-kB, INF-γ, and iNOS and a decrease in IL-10, IL-22, and IL-10R expression after the FUC treatment of injured liver rats. For the hepatic inflammation and PAC-toxicity-induced oxidative stress genes and proteins, FUC-treated rats (100, 200, and 500 mg/kg) showed a reduction in the expression of oxidative stress genes. These results showed that FUC protected the liver against PAC-induced injury through antioxidant and anti-inflammatory actions. However, further clinical studies are required to confirm the findings.

Vitamin D and calcium co-therapy mitigates pre-established cadmium nephropathy by regulating renal calcium homeostatic molecules and improving anti-oxidative and anti-inflammatory activities in rat.

BACKGROUND: Cadmium (Cd) is a major environmental pollutant and chronic toxicity could induce nephropathy by increasing renal oxidative stress and inflammation. Although vitamin D (VD) and calcium (Ca) prophylactic treatments attenuated Cd-induced cell injury, none of the prior studies measure their renoprotective effects against pre-established Cd-nephropathy. AIMS: To measure the alleviating effects of VD and/or Ca single and dual therapies against pre-established nephrotoxicity induced by chronic Cd toxicity prior to treatment initiation. METHODS: Forty male adult rats were allocated into: negative controls (NC), positive controls (PC), Ca, VD and VC groups. The study lasted for eight weeks and all animals, except the NC, received CdCl2 in drinking water (44 mg/L) throughout the study. Ca (100 mg/kg) and/or VD (350 IU/kg) were given (five times/week) during the last four weeks to the designated groups. Subsequently, the expression of transforming growth factor-ß (TGF-ß1), inducible nitric oxide synthase (iNOS), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), VD synthesising (Cyp27b1) and catabolizing (Cyp24a1) enzymes with VD receptor (VDR) and binding protein (VDBP) was measured in renal tissues. Similarly, renal expression of Ca voltage-dependent channels (CaV1.1/CaV3.1), store-operated channels (RyR1/ITPR1), and binding proteins (CAM/CAMKIIA/S100A1/S100B) were measured. Serum markers of renal function alongside several markers of oxidative stress (MDA/H2O2/GSH/GPx/CAT) and inflammation (IL-6/TNF-α/IL-10) together with renal cell apoptosis and expression of caspase-3 were also measured. RESULTS: The PC group exhibited hypovitaminosis D, hypocalcaemia, hypercalciuria, proteinuria, reduced creatinine clearance, and increased renal apoptosis/necrosis with higher caspase-3 expression. Markers of renal tissue damage (TGF-ß1/iNOS/NGAL/KIM-1), oxidative stress (MDA/H2O2), and inflammation (TNF-α/IL-1ß/IL-6) increased, whilst the antioxidants (GSH/GPx/CAT) and IL-10 decreased, in the PC group. The PC renal tissues also showed abnormal expression of Cyp27b1, Cyp24a1, VDR, and VDBP, alongside Ca-membranous (CaV1.1/CaV3.1) and store-operated channels (RyR1/ITPR1) and cytosolic Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B). Although VD was superior to Ca monotherapy, their combination revealed the best mitigation effects by attenuating serum and renal tissue Cd concentrations, inflammation and oxidative stress, alongside modulating the expression of VD/Ca-molecules. CONCLUSIONS: This study is the first to show improved alleviations against Cd-nephropathy by co-supplementing VD and Ca, possibly by better regulation of Ca-dependent anti-oxidative and anti-inflammatory actions.

Cymbopogon citratus and Citral Overcome Doxorubicin Resistance in Cancer Cells via Modulating the Drug's Metabolism, Toxicity, and Multidrug Transporters.

Multidrug resistance (MDR) is the major complex mechanism that causes the failure of chemotherapy, especially with drugs of natural origin such as doxorubicin (DOX). Intracellular drug accumulation and detoxification are also involved in cancer resistance by reducing the susceptibility of cancer cells to death. This research aims to identify the volatile composition of Cymbopogon citratus (lemon grass; LG) essential oil and compare the ability of LG and its major compound, citral, to modulate MDR in resistant cell lines. The composition of LG essential oil was identified using gas chromatography mass spectrometry (GC-MS). In addition, a comparison of the modulatory effects of LG and citral, performed on breast (MCF-7/ADR), hepatic (HepG-2/ADR), and ovarian (SKOV-3/ADR) MDR cell lines, were compared to their parent sensitive cells using the MTT assay, ABC transporter function assays, and RT-PCR. Oxygenated monoterpenes (53.69%), sesquiterpene hydrocarbons (19.19%), and oxygenated sesquiterpenes (13.79%) made up the yield of LG essential oil. α-citral (18.50%), ß-citral (10.15%), geranyl acetate (9.65%), ylangene (5.70), δ-elemene (5.38%), and eugenol (4.77) represent the major constituents of LG oil. LG and citral (20 µg/mL) synergistically increased DOX cytotoxicity and lowered DOX dosage by >3-fold and >1.5-fold, respectively. These combinations showed synergism in the isobologram and CI < 1. DOX accumulation or reversal experiment confirmed that LG and citral modulated the efflux pump function. Both substances significantly increased DOX accumulation in resistant cells compared to untreated cells and verapamil (the positive control). RT-PCR confirmed that LG and citral targeted metabolic molecules in resistant cells and significantly downregulated PXR, CYP3A4, GST, MDR1, MRP1, and PCRP genes. Our results suggest a novel dietary and therapeutic strategy combining LG and citral with DOX to overcome multidrug resistance in cancer cells. However, these results should be confirmed by additional animal experiments before being used in human clinical trials.

In Vitro Anticancer and Antibacterial Activities of the Essential Oil of Forsskal's Basil Growing in Extreme Environmental Conditions.

Many species belonging to the genus Ocimum are used for aromatic, medicinal, and cosmetic purposes. The essential oil (OFEO) obtained by hydrodistillation of the flowering aerial parts of Forsskal's Basil "Ocimum forskolei Benth" growing in extreme environmental conditions in Mecca Region, Saudi Arabia was analyzed by GC-MS. The main constituents were phenylpropanoids (methyl eugenol 55.65% and eugenol 11.66%), monoterpene (linalool 9.75%), and sesquiterpenes (germacrene D 3.72% and ß-caryophyllene 2.57%). The OFEO was tested against MCF7, HT29, and HCT116 cancer cells and compared with normal fibroblast cells (MRC5). The MTT assay showed that HCT116 was more sensitive to OFEO (IC50 5.34 µg/mL), which reduced the number of HCT116 colonies at 6 µg/mL, while causing complete colony death at 12 and 24 µg/mL. Western Blotting and qRT-PCR were used to evaluate the level change of different proteins with respect to GAPDH. OFEO upregulated the apoptotic protein (caspase 3), and downregulated the cell proliferation proteins (AKT and pAKT), cell cycle arrest (PCNA, Cyclin D1), and the anti-apoptotic Bcl2 proteins. OFEO was also tested against reference strains of Gram-negative and Gram-positive bacteria including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, and Staphylococcus aureus by using the well-diffusion and assessing their MICs, which ranged from 250 to 500 µg/mL.

Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells.

This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

Evaluation of the Anti-Obesity Effect of Zeaxanthin and Exercise in HFD-Induced Obese Rats.

Obesity is a worldwide epidemic associated with many health problems. One of the new trends in health care is the emphasis on regular exercise and a healthy diet. Zeaxanthin (Zea) is a carotenoid with many beneficial effects on human health. The aim of this study was to investigate whether the combination of Zea and exercise had therapeutic effects on obesity induced by an HFD in rats. Sixty male Wistar rats were randomly divided into five groups of twelve: rats fed a standard diet; rats fed a high-fat diet (HFD); rats fed an HFD with Zea; rats fed an HFD with Exc; and rats fed an HFD with both Zea and Exc. To induce obesity, rats were fed an HFD for twelve weeks. Then, Zea and exercise were introduced with the HFD for five weeks. The results showed that the HFD significantly increased visceral adipose tissue, oxidative stress, and inflammation biomarkers and reduced insulin, high-density lipoprotein, and antioxidant parameters. Treatments with Zea, Exc, and Zea plus Exc reduced body weight gain, triacylglycerol, glucose, total cholesterol, and nitric oxide levels and significantly increased catalase and insulin compared with the HFD group. This study demonstrated that Zea administration and Exc performance appeared to effectively alleviate the metabolic alterations induced by an HFD. Furthermore, Zea and Exc together had a better effect than either intervention alone.

Enhanced anti-cancer effects of oestrogen and progesterone co-therapy against colorectal cancer in males.

Although ovarian sex steroids could have protective roles against colorectal cancer (CRC) in women, little is currently known about their potential anti-tumorigenic effects in men. Hence, this study measured the therapeutic effects of 17ß-oestradiol (E2) and/or progesterone (P4) against azoxymethane-induced CRC in male mice that were divided into (n = 10 mice/group): negative (NC) and positive (PC) controls, E2 (580 µg/Kg/day; five times/week) and P4 (2.9 mg/Kg/day; five times/week) monotherapies, and concurrent (EP) and sequential (E/P) co-therapy groups. Both hormones were injected intraperitoneally to the designated groups for four consecutive weeks. Similar treatment protocols with E2 (10 nM) and/or P4 (20 nM) were also used in the SW480 and SW620 human male CRC cell lines. The PC group showed abundant colonic tumours alongside increased colonic tissue testosterone levels and androgen (AR) and oestrogen (ERα) receptors, whereas E2 and P4 levels with ERß and progesterone receptor (PGR) decreased significantly compared with the NC group. E2 and P4 monotherapies equally increased ERß/PGR with p21/Cytochrome-C/Caspase-3, reduced testosterone levels, inhibited ERα/AR and CCND1/survivin and promoted apoptosis relative to the PC group. Both co-therapy protocols also revealed better anti-cancer effects with enhanced modulation of colonic sex steroid hormones and their receptors, with E/P the most prominent protocol. In vitro, E/P regimen showed the highest increases in the numbers of SW480 (2.1-fold) and SW620 (3.5-fold) cells in Sub-G1 phase of cell cycle. The E/P co-therapy also disclosed the lowest percentages of viable SW480 cells (2.8-fold), whilst both co-therapy protocols equally showed the greatest SW620 apoptotic cell numbers (5.2-fold) relative to untreated cells. Moreover, both co-therapy regimens revealed maximal inhibitions of cell cycle inducers, cell survival markers, and AR/ERα alongside the highest expression of cell cycle suppressors, pro-apoptotic molecules, and ERß/PGR in both cell lines. In conclusion, CRC was associated with abnormal levels of colonic sex steroid hormones alongside aberrant protein expression of their receptors. While the anti-cancer effects of E2 and P4 monotherapies were equal, their combination protocols showed boosted tumoricidal actions against CRC in males, possibly by promoting ERß and PGR-mediated androgen deprivation together with inhibition of ERα-regulated oncogenic pathways.

Enhanced in vitro tumoricidal effects of 5-Fluorouracil, thymoquinone, and active vitamin D3 triple therapy against colon cancer cells by attenuating the PI3K/AKT/mTOR pathway.

AIMS: This study measured the effects of 5-Fluorouracil (5-FU), calcitriol (VD3), and/or thymoquinone (TQ) single/dual/triple therapies on cell cycle progression, apoptosis, inhibition of the PI3K/AKT/mTOR pathway, and oxidative stress against colorectal cancer (CRC). MAIN METHODS: The HT29, SW480 and SW620 cell lines were treated with 5-FU (50 µM), VD3 (25 µM), and TQ (75 µM), alone or combined for 12 h, prior to cell cycle/apoptosis analyses. KEY FINDINGS: TQ monotherapy had greater anticancer effects to active VD3 or 5-FU, revealing higher expression of p21/p27/PTEN/BAX/Cyto-C/Casp-3 and increased levels of total glutathione, with inhibitions in CCND1/CCND3/BCL-2 and PI3K/AKT/mTOR molecules, alongside higher rates of apoptosis in HT29, SW480 and SW620 cells (P < 0.005 for all markers). Additionally, all combination protocols revealed enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway, higher expression of p21/p27/PTEN/BAX/Cyto-C/Casp-3, and better anti-oxidant effects, than the monotherapies. Although TQ/5-FU and TQ/VD3 co-therapies were better relative to the VD3/5-FU regimen, the best tumoricidal effects were observed with triple therapy in the HT29 and SW480 cell lines, possibly by boosted attenuations of the PI3K/AKT/mTOR oncogenic pathway. In contrast, TQ single treatment was more effective than the triple therapy regimen in metastatic SW620 cells, suggesting that this protocol would be more useful therapeutically in late-stage CRC. SIGNIFICANCE: In conclusion, this study is the first to demonstrated enhanced anti-tumorigenic effects for VD3, TQ, and 5-FU triple therapy against CRC cells and could represent the best strategy for treating early stages of malignancy, whereas TQ monotherapy could be a better approach for treating metastatic forms of the disease.

Beta 2 microglobulin correlates with oxidative stress in elderly.

Beta 2 microglobulin (Β2M) is expressed in all nucleated cells, it interplays with mediators to regulate and modulate cellular functions. Its role in aging associated disorders has been documented recently. Oxidative stress has been known to play a direct implication on these disorders. Therefore, there is a rationality to explore the function of Β2M in oxidative stress in elderly people. The aim of the study was to assess the Β2M levels in different group of age, and to study the correlation between Β2M and oxidative stress. Actually, the serum levels of Β2M increased significantly in old people comparing to youngers. In addition, there was a positive correlation between Β2M levels and the age of participants (p < 0.001). In addition, there was a positive correlation between Β2M levels and Malondialdehyde (MDA) (p < 0.001), which underscored the possible role of Β2M in oxidative stress. To confirm the previous result, the correlation between total antioxidant capacity (TAC) and Β2M was assessed. There was a negative correlation between them (p < 0.001). These results suggested a possible role of Β2M in oxidative stress status in elderly people; in addition, it suggested the ability of using Β2M as a novel biomarker for oxidative stress. However, further work should be conducted to explore the exact role of Β2M in oxidative stress, and to include large sample size to confirm the results before translating the findings to clinic.

Vitamin D protects against oxidative stress, inflammation and hepatorenal damage induced by acute paracetamol toxicity in rat.

Acute paracetamol (APAP) toxicity is a leading cause of liver, and less commonly renal, injuries through oxidative stress and inflammation. Albeit vitamin D (VD) is a well-known anti-oxidant and anti-inflammatory hormone, there is no report on its potential protective/therapeutic actions against APAP acute toxicity. This study, therefore, measured the interplay between APAP toxicity and the hepatorenal expressions of the VD-metabolising enzymes (Cyp2R1, Cyp27b1 & cyp24a1), receptor (VDR) and binding protein (VDBP) alongside the effects of VD treatment on APAP-induced hepatorenal injuries. Thirty-two male rats were distributed equally into negative (NC) and positive (PC) controls besides VD prophylactic (P-VD) and therapeutic (T-VD) groups. All groups, except the NC, received a single oral dose of APAP (1200 mg/kg). The P-VD also received by intraperitoneal injection two cycles of VD3 (1000 IU/Kg/day; 5 days/week) prior to, and a third round after, APAP administration. Similarly, the T-VD group received VD3 (3000 IU/Kg/day) for five successive days post-APAP intoxication. Euthanasia was on the sixth day post-APAP toxicity. The PC group had marked alterations in the hepatorenal biochemical parameters, upregulation in cellular cleaved caspase-3 as well as pronounced increase in the numbers of apoptotic/necrotic cells by TUNEL technique. The PC group plasma levels of 25-hydroxyvitamin D (25-OH VD) also declined markedly and coincided with significant inhibitions in the expression of Cyp2R1 and Cyp27b1 enzymes and VDR, whereas the VDBP and Cyp24a1 increased substantially, in the hepatorenal tissues at the gene and protein levels compared with the NC group. Coherently, the lipid peroxidation marker (MDA) and pro-inflammatory cytokines (IL1ß, IL6, IL17A, IFN-γ & TNF-α) augmented significantly, while the anti-oxidative markers (GSH, GPx & CAT) and anti-inflammatory cytokines (IL10 & IL22) diminished substantially, in the PC hepatorenal tissues. Both VD regimens alleviated the APAP-induced hepatorenal damages and restored the 25-OH VD levels together with the hepatorenal expression of Cyp2R1, Cyp27b1, Cyp24a1, VDR and VDBP. Additionally, MDA and all the targeted pro-inflammatory cytokines declined, whereas all the anti-oxidative and anti-inflammatory markers increased, in both VD groups hepatorenal tissues and the results were significantly different than the PC group. Although the P-VD anti-inflammatory and anti-oxidative stress actions were more pronounced than the T-VD group, the results remained markedly abnormal than the NC group. In conclusion, this report is the first to reveal that the circulatory VD levels alongside the hepatorenal VD-metabolising enzymes and VDR are pathologically altered following acute APAP toxicity. Moreover, the prophylactic protocol showed better anti-oxidative and anti-inflammatory effects than the therapeutic regimen against APAP-induced hepatorenal injuries.

Assessment and management of mercury poisoning with an unusual abdominal x-ray!

This case discusses a gentleman who presented to the medical team after ingesting mercury liquid. We discuss the assessment, management and different types of mercury poisoning.