Doxorubicin-sanguinarine nanoparticles: formulation and evaluation of breast cancer cell apoptosis and cell cycle.

BACKGROUND: Therapeutic resistance fails cancer treatment. Drug-nanoparticle combinations overcome resistance. Sanguinarine-conjugated nanoparticles may boost sanguinarine's anticancer effects. METHODS: Sanguinarine, HPMC-NPs, and doxorubicin were tested on Adriamycin-resistant MCF-7/ADR breast cancer cells, parent-sensitive MCF-7, and MCR-5 normal cells (DX). RESULTS: Regular distribution, 156 nm diameter, <1 µm average size, 100% intensity-SN is therapeutic. Furthermore, the obtained NPs showed PDI = 0.145, zeta-potential=-37.6, and EE%=90.5%. DX sensitized MCF-7 cells (IC50 = 1.4 µM) more than MCF-7/ADR cells (IC50 = 27 µM) with RR = 19.3. SA and SN were more toxic to MCF-7/ADR cells (overexpressed with P-gp) than their sensitive parent MCF-7 cells (IC50 = 4 µM, RR = 0.6 and 0.6 µM, RR = 0.7). MCR-5 normal lung cells were more resistant to SA (IC50 = 7.2 µM) and SN (IC50 = 1.6 µM) with a selection index > 2. Synergistic cytotoxic interactions reduced the IC50 from 27 µM to 1.6 (CI = 0.1) and 0.9 (CI = 0.4) after DX and nontoxic dosages (IC20) of SA and SN. DS and SN killed 27.1% and 39.4% more cells than DX (7.7%), SA (4.9%), SN (5.5%), or untreated control (0.3%). DS and DSN lowered CCND1 and survival in MCF-7/ADR cells while raising p21 and Casp3 gene and protein expression. CONCLUSIONS: Cellular and molecular studies suggested adjuvant chemosensitizers SA and SN to reverse MDR in breast cancer cells.

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.

Patterns of acute poisoning for children during outbreak of Corona virus in Makkah region Saudi Arabia.

Background: Poisoning occurs when a person is exposed to an external substance at a too high dose for them. It is possible for young children to be exposed to chemicals. Lungs, the heart, CNS, the digestive tract, and kidneys can be poisoned. In 2004, over 45,000 children and teenagers died from acute poisoning, representing 13% of all accidental poisoning deaths worldwide. Poisoning patterns vary by exposure type, age group, poison type, and dose. Aim: This study assessed the pattern of acute poisoning with drugs, chemicals, and natural toxins among children (<12 years old). The study was done in Makkah region and registered in the poison control center in Makkah, the forensic chemistry center in Haddah during 2020-2021. Methods: A retrospective cohort study was done on 122 children exposed to toxic substances in Makkah. The children were 12 years old and had good health for a maximum of one year. Stratified random sampling was used to divide cases into groups of similar poisons (pharmaceutical products, household products, plant envenomation, and animal envenomation). Then each group got a random samples. The data were analysed with SPSS software. Results: The mean age of children was 5.2 years, with 59% being boys. The mean temperature, pulse, systolic, diastolic, and respiratory rates were 36.77, 98.29, 109.1, 69.17, and 21.49. The most documented pharmaceutical products (200 mg) were carbamazepine (5 mg), methanol, risperidone (5 mg), propranolol (5 mg), and olanzapine (5 mg). The most common poison forms were tablets (42.6%), syrups (15.6%), capsules (13.9%), and solutions (13.1%). The most common poisoning routes were ingestion (82.8%), dermal (5.7%), injection (4.9%), and inhalation (6.6%). Accidental poisoning was 83%, with a 30-minute lag for 30.3% of children, and most (69.7%) occurred at home. Benzodiazepines were the most commonly used category class drug (18%), with normal pupils and an ECG of 85.2%. Sixty-seven percent had blood tests. Sickness was 9.48, and the positive result was 213.01. The most common presenting symptoms were GIT and neurological (23.8%). 31.1% had mild, moderate, or severe toxicity. Most cases (68%) were complex. 34.4% were intubated, 9.8% had repeated-dose-activated charcoal for enhanced elimination, and 27.8% were on IV fluids. Children with GIT, CVS, respiratory, dermal, and neurological symptoms had a higher percentage of severe toxicity (p < 0.05). Slight toxicity was associated with whole bowel irrigation, intubation for oxygen therapy, N-acetylcysteine or sedation, fluids, and phenytoin (P < 0.05). Complicated cases had a higher mean AST/IUL than non-complicated cases (75.5 vs. 20.08, p < 0.05). The level of toxicity did not correlate with the mean of all lab tests (p > 0.05). The age of the children correlated positively with their systolic BP (r = 0.22, p < 0.01). Conclusion: The results show how important it is to teach the public about poisoning and make rules for tracking and dealing with poisonings in Saudi Arabia.

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.

Liver Injury in Favipiravir-Treated COVID-19 Patients: Retrospective Single-Center Cohort Study.

(1) Background: Favipiravir (FVP) is a new antiviral drug used to treat COVID-19. It has been authorized to be used in the kingdom of Saudi Arabia in the treatment of COVID-19. The mechanism of action of FVP is working as a specific inhibitor for the RNA-dependent RNA polymerase of the RNA chain virus. FVP has the potential to be hepatotoxic because of the structure similarity with pyrazinamide. This retrospective study aimed to determine the prevalence of liver injury in FVP-treated COVID-19 patients in General East Jeddah Hospital, Saudi Arabia, during the COVID-19 pandemic. (2) Methods: A total of 6000 patients infected with COVID-19 and treated at the East Jeddah Hospital were included, with a sample size of 362 patients. The participants ranged from 18 to 70 years of age, both males and females, with normal hepatic and renal function and had a confirmed diagnosis of COVID-19 infection. Patients who had gouty arthritis, hepatic and renal dysfunction, dead patients, pregnant women, and breastfeeding mothers were all excluded from this study. A retrospective cohort study compared two groups of patients treated with and without FVP and who followed the Saudi Ministry of Health protocol to manage COVID-19 infection. (3) Results: An adverse effect of FVP on the liver was found that ranged from mild to severe. Stopping treatment with FVP was associated with an observed important increase in the levels of liver enzymes AST (p < 0.001), ALT (p < 0.001), alkaline phosphatase (p < 0.03), total bilirubin (p < 0.001), and direct bilirubin (p < 0.001) in the treated compared with the untreated group. (4) Conclusion: This study showed a significant difference between the treated and the untreated groups with FVP in liver injury. FVP influences the liver, increasing the blood levels of the liver function parameters.

The use of the nutritional supplements during the covid-19 outbreak in Saudi Arabia: A cross-sectional study.

BACKGROUND: COVID-19 causes moderate to severe illness and is spreading globally. During a pandemic, vitamins and minerals are vital to health. Therefore, the prevalence and epidemiology of supplement use in Saudi Arabia during the COVID-19 pandemic must be known. METHODS: This cross-sectional study was conducted in Saudi Arabia using an online survey. The study was conducted from June to March 2022 on both adults and children. The link to the survey was shared on social media platforms. The survey included questions on participants' demographics, vaccination status, supplements they used, and side effects of supplements. Participation in this study was optional, and there was no obligation to participate. There was a declaration about the aim of the study and different objectives before starting the survey. RESULTS: The present study reported that most of the participants reported that they used vitamin C (64.6 %), zinc (51.9 %), multivitamins (46.1 %), black seeds (26.7 %), garlic (Allium sativum) (15.4 %), omega-3 (22.1 %), vitamin D (22.2 %), echinacea (10.1 %), manuka honey (26.0 %), curcumin (13.6 %), ginger (22.5 %), royal jelly (12.9 %), and propolis (7.5 %) before and during the COVID-19 pandemic period. These supplements were used more frequently by subjects during the pandemic than before. DISCUSSION AND CONCLUSION: The respondents' risk of these supplements' use may partially reflect the public's behavioral response during a pandemic. Future studies can document the health beliefs and motivations of nutritional supplement users.

In Vivo and In Vitro Enhanced Tumoricidal Effects of Metformin, Active Vitamin D3, and 5-Fluorouracil Triple Therapy against Colon Cancer by Modulating the PI3K/Akt/PTEN/mTOR Network.

Chemoresistance to 5-fluorouracil (5-FU) is common during colorectal cancer (CRC) treatment. This study measured the chemotherapeutic effects of 5-FU, active vitamin D3 (VD3), and/or metformin single/dual/triple regimens as complementary/alternative therapies. Ninety male mice were divided into: negative and positive (PC) controls, and 5-FU, VD3, Met, 5-FU/VD3, 5-FU/Met, VD3/Met, and 5-FU/VD3/Met groups. Treatments lasted four weeks following CRC induction by azoxymethane. Similar regimens were also applied in the SW480 and SW620 CRC cell lines. The PC mice had abundant tumours, markedly elevated proliferation markers (survivin/CCND1) and PI3K/Akt/mTOR, and reduced p21/PTEN/cytochrome C/caspase-3 and apoptosis. All therapies reduced tumour numbers, with 5-FU/VD3/Met being the most efficacious regimen. All protocols decreased cell proliferation markers, inhibited PI3K/Akt/mTOR molecules, and increased proapoptotic molecules with an apoptosis index, and 5-FU/VD3/Met revealed the strongest effects. In vitro, all therapies equally induced G1 phase arrest in SW480 cells, whereas metformin-alone showed maximal SW620 cell numbers in the G0/G1 phase. 5-FU/Met co-therapy also showed the highest apoptotic SW480 cell numbers (13%), whilst 5-FU/VD3/Met disclosed the lowest viable SW620 cell percentages (81%). Moreover, 5-FU/VD3/Met revealed maximal inhibitions of cell cycle inducers (CCND1/CCND3), cell survival (BCL2), and the PI3K/Akt/mTOR molecules alongside the highest expression of cell cycle inhibitors (p21/p27), proapoptotic markers (BAX/cytochrome C/caspase-3), and PTEN in both cell lines. In conclusion, metformin monotherapy was superior to VD3, whereas the 5-FU/Met protocol showed better anticancer effects relative to the other dual therapies. However, the 5-FU/VD3/Met approach displayed the best in vivo and in vitro tumoricidal effects related to cell cycle arrest and apoptosis, justifiably by enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway.

Sage, Salvia officinalis L., Constituents, Hepatoprotective Activity, and Cytotoxicity Evaluations of the Essential Oils Obtained from Fresh and Differently Timed Dried Herbs: A Comparative Analysis.

Sage, Salvia officinalis L., is used worldwide as an aromatic herb for culinary purposes as well as a traditional medicinal agent for various ailments. Current investigations exhibited the effects of extended dryings of the herb on the yields, composition, oil quality, and hepatoprotective as well as anti-cancer biological activities of the hydrodistillation-obtained essential oils from the aerial parts of the plant. The essential oils' yields, compositions, and biological activities levels of the fresh and differently timed and room-temperature dried herbs differed significantly. The lowest yields of the essential oil were obtained from the fresh herbs (FH, 631 mg, 0.16%), while the highest yield was obtained from the two-week dried herbs (2WDH, 1102 mg, 0.28%). A notable decrease in monoterpenes, with increment in the sesquiterpene constituents, was observed for the FH-based essential oil as compared to all the other batches of the essential oils obtained from the different-timed dried herbs. Additionally, characteristic chemotypic constituents of sage, i.e., α-pinene, camphene, ß-pinene, myrcene, 1, 8-cineole, α-thujone, and camphor, were present in significantly higher proportions in all the dried herbs' essential oils as compared to the FH-based essential oil. The in vivo hepatoprotective activity demonstrated significant reductions in the levels of AST, ALT, and ALP, as well as a significant increase in the total protein (p < 0.05) contents level, as compared to the acetaminophen (AAP) administered experimental group of rats. A significant reduction (p < 0.05) in the ALT level was demonstrated by the 4WDH-based essential oil in comparison to the FH-based essential oil. The levels of creatinine, cholesterol, and triglycerides were reduced (p < 0.05) in the pre-treated rats by the essential oil batches, with non-significant differences found among them as a result of the herbs dryings based oils. A notable increase in the viability of the cells, and total antioxidant capacity (TAOxC) levels, together with the reduction in malondialdehyde (MDA) levels were observed by the essential oils obtained from all the batches as compared with the AAP-treated cell-lines, HepG-2, HeLa, and MCF-7, that indicated the in vitro hepatoprotective effects of the sage essential oils. However, significant improvements in the in vivo and in vitro hepatoprotective activities with the 4WDH-based oil, as compared to all other essential oil-batches and silymarin standard demonstrated the beneficial effects of the drying protocol for the herb for its medicinal purposes.

Multiple Molecular Mechanisms to Overcome Multidrug Resistance in Cancer by Natural Secondary Metabolites.

Plant secondary metabolites (SMs) common natural occurrences and the significantly lower toxicities of many SM have led to the approaching development and use of these compounds as effective pharmaceutical agents; especially in cancer therapy. A combination of two or three of plant secondary metabolites together or of one SM with specific anticancer drugs, may synergistically decrease the doses needed, widen the chemotherapeutic window, mediate more effective cell growth inhibition, and avoid the side effects of high drug concentrations. In mixtures they can exert additive or even synergistic activities. Many SM can effectively increase the sensitivity of cancer cells to chemotherapy. In phytotherapy, secondary metabolites (SM) of medicinal plants can interact with single or multiple targets. The multi-molecular mechanisms of plant secondary metabolites to overcome multidrug resistance (MDR) are highlighted in this review. These mechanisms include interaction with membrane proteins such as P-glycoprotein (P-gp/MDR1); an ATP-binding cassette (ABC) transporter, nucleic acids (DNA, RNA), and induction of apoptosis. P-gp plays an important role in the development of MDR in cancer cells and is involved in potential chemotherapy failure. Therefore, the ingestion of dietary supplements, food or beverages containing secondary metabolites e.g., polyphenols or terpenoids may alter the bioavailability, therapeutic efficacy and safety of the drugs that are P-gp substrates.

Evaluation of the Diagnostic and Predicative Values of 8-Iso-Prostaglandin F2α as a Biomarker of Breast Cancer.

BACKGROUND: Breast cancer (BC) is a commonly reported cancer that is widely prevalent among women. Its early detection improves patient survival and results in better outcomes. For diagnosis and follow-up care, tumor markers are one of the feasible investigations to be ordered. 8-Iso-prostaglandin F2α (8-iso-PGF2α) serves as a serum marker reflecting oxidative stress and subsequent damaging of DNA. In the present study, we aimed to evaluate both diagnostic and predictive values of 8-iso-PGF2α in BC patients. MATERIALS AND METHODS: Serum levels of 8-iso-PGF2α were assessed for 66 women with benign breast tumors and 65 women who had malignant BC. To compare the patients who had breast tumors with healthy individuals, 63 women free of breast diseases were selected as controls. RESULTS: The serum level of 8-iso-PGF2α in the BC patients (57.92 pg/mL) was significantly higher compared to those with benign tumors (18.89 pg/mL) (p < 0.001). In addition, individuals with no breast diseases had less 8-iso-PGF2α (4.02 pg/mL) compared to those who had developed a tumor (p < 0.001). Serum 8-iso-PGF2α was found to be positively correlated with both carcinoembryonic antigen (r = 0.74, p < 0.001) and cancer antigen 15-3 (r = 0.80, p < 0.001). Furthermore, serum 8-iso-PGF2α showed high diagnostic performance in BC (AUC = 0.999, sensitivity = 100%, specificity = 99.2% at a cutoff value of 36.18 pg/mL). CONCLUSIONS: Our study found that the high level of serum 8-iso-PGF2α helps to provide a non-invasive indicator to detect BC. Future work with a larger sample size and various phases of BC can confirm the current results which provide insights into the early detection of cancer.

The carotenoid fucoxanthin can sensitize multidrug resistant cancer cells to doxorubicin via induction of apoptosis, inhibition of multidrug resistance proteins and metabolic enzymes.

BACKGROUND: Multidrug resistance (MDR) causes failure of doxorubicin therapy of cancer cells, which develops after or during doxorubicin treatment resulting in cross-resistance to structurally and functionally-unrelated other anticancer drugs. MDR is multifactorial phenomenon associated with overexpression of ATP-binding cassette (ABC) transporters, metabolic enzymes, impairment of apoptosis, and alteration of cell cycle checkpoints. The cancer-prevention of the dietary carotenoid; fucoxanthin (FUC) has been extensively explored. Nevertheless, the underlying mechanism of its action is not full elucidated. HYPOTHESIS/PURPOSE: Investigation of the underlying mechanism of MDR reversal by the dietary carotenoid fucoxanthin (FUC) and its ability to enhance the doxorubicin (DOX) cytotoxicity in resistant breast (MCF-7/ADR), hepatic (HepG-2/ADR), and ovarian (SKOV-3/ADR) cell lines. METHODS: The synergistic interaction of FUC and DOX was evaluated using several techniques, viz.; MTT assay, ABC transporter function assays using FACS and fluorimetry, enzyme activity via spectroscopy and luminescence assays, and apoptosis assay using FACS, and gene expression using RTPCR. RESULTS: FUC (20 µM) synergistically enhanced the cytotoxicity of DOX and significantly reduced the dose of DOX (FR) in DOX resistant cells (MCF-7/ADR), hepatic (HepG-2/ADR), and ovarian (SKOV-3/ADR) to 8.42-(CI= 0.25), 6.28-(CI= 0.32), and 4.56-fold (CI=0.37) (P<0.001). FUC significantly increased the accumulation of DOX more than verapamil in resistant cells by 2.70, 2.67, and 3.95-fold of untreated cells (p<0.001), respectively. A FUC and DOX combination significantly increased the Rho123 accumulation higher than individual drugs by 2.36-, 2.38-, 1.89-fold verapamil effects in tested cells (p<0.001), respectively. The combination of the FUC and DOX decreased ABCC1, ABCG2, and ABCB1 expression. The FUC and DOX combination increased the levels and activity of caspases (CASP3, CASP8) and p53, while decreased the levels and activity of CYP3A4, GST, and PXR in resistant cancer cells. The combination induced early/late apoptosis to 91.9/5.4% compared with 0.0/0.7% of untreated control. CONCLUSION: Our data suggests a new dietary and therapeutic approach of combining the FUC with DOX to overcome multidrug resistance in cancer cells. However, animal experiments should be conducted to confirm the findings before applying the results into clinical trials.

BTK targeting suppresses inflammatory genes and ameliorates insulin resistance.

Type 2 diabetes (T2D) causes profound psychological and physical distress to patients and burdens the health-care system. Although several antidiabetic drugs have been approved, none of them are adequately effective in the long-term management of T2D. Therefore, novel treatment options are needed for disease prevention or delaying disease progression. Bruton's tyrosine kinase (BTK) is a cytoplasmic enzyme that plays a role in B-cell differentiation and proliferation, and therapeutic targeting of BTK offers protection against chronic diseases. In this study, we analyzed BTK expression and its correlation with inflammatory mediators in patients with diabetes and obesity. The levels of BTK were significantly high in visceral adipose tissues of patients (p < 0.01) with diabetes and obesity compared with healthy controls. Additionally, a positive correlation was noted between the expression of BTK and the inflammatory cytokine genes TNF-α, INF-γ, IL-6, and IL-1 (p < 0.01) in adipose tissue. In insulin-resistant HepG2 cells (IR-HepG2), ibrutinib inhibited BTK expression in parallel with inflammatory genes, and increased insulin signaling and activity compared with untreated IR-HepG2 cells. Additionally, ibrutinib-treated IR-HepG2 cells showed increased glucose uptake compared with untreated IR-HepG2 cells. These results provide evidence that BTK inhibition may serve as a novel therapeutic strategy for the treatment of T2D. These findings also uncover the novel role of BTK in diabetes and insulin resistance; however, further in vivo studies are required prior to translating the findings into clinical settings.

Overexpression of Inducible Nitric Oxide Synthase in Allergic and Nonallergic Nasal Polyp.

Sinonasal polyps are very common benign lesions of the nasal mucosa. Most of nasal polyps (NP) are idiopathic, and the pathophysiology of this disease is still incompletely understood. Nitric oxide (NO) is a reactive molecule generated by nitric oxide synthase (NOS). NO has been identified as an important mediator in airway function and pathogenesis of several respiratory system diseases. Histological and genetical expression of iNOS was detected to evaluate the role of NO in the pathogenesis of allergic (ANP) and nonallergic nasal polyps (NANP). Forty patients with nasal polyps (20 allergic and 20 nonallergic) were identified by history, clinical examination, and investigation. NPs were obtained from the middle turbinate (MT) during concha bullosa surgery. Twenty normal MT nasal tissues were taken as the control from patients undergoing concha bullosa surgery, without any evidence of allergy or inflammation. A nasal polyp specimen from each patient was subjected for immune-histochemical study followed by histological examination to detect the expression of iNOS. RT-PCR was used to evaluate the iNOS gene expression in isolated tissues. The expression of iNOS in both epithelial and stromal layers was greater in NP than in MT tissues. The ANP group showed more iNOS expression than those of the NANP group. The relative mRNA levels of iNOS gene were significantly higher in ANP (2.5-fold) compared to the normal (1.02-fold, P < 0.001) and NANP (1.5-fold, P < 0.01) groups. NP exhibited a significantly high expression of iNOS at both histological and genetical levels. NO might be an essential factor in the life history of NP. Further studies in a larger sample size are required to explain the probable mechanisms of NO in pathogenesis of NP.

8-Hydroxy-2'-deoxyguanosine as a Discriminatory Biomarker for Early Detection of Breast Cancer.

BACKGROUND: Breast cancer (BC) is one of the most prevalent and reported cancers among Saudi women. Detection of BC in the early invasive stage (stages I, II) has an advantage in treating patients over detection in the late invasive stage (stages III, IV). Tumor markers are used to aid in diagnosis, treatment monitoring, and recurrence detection of malignant tumors. 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a marker of nucleic damage owing to oxidative stress. PATIENTS AND METHODS: We studied the blood levels of 8-OHdG in 50 women with benign breast tumors, 50 women with BC, and 50 healthy women as a control group. RESULTS: The concentrations of 8-OHdG were significantly increased in the BC group (55.2 ng/dL) compared with the benign tumor group (30.2 ng/dL) and with the healthy control group (9.08 ng/dL). The same pattern was observed with other diagnostic markers, including carcinoembryonic antigen and cancer antigen 15-3. Significant positive correlations between 8-OHdG and both carcinoembryonic antigen (r = 0.63; P < .001) and cancer antigen 15-3 (r = 0.51; P < .001) were noticed. The levels of 8-OHdG were significantly higher in stage I (81 ng/dL) compared with stage II (51 ng/dL; P < .05), stage III (38 ng/dL; P < .01), and stage IV (19 ng/dL; P < .001). In addition, serum 8-OHdG had a high diagnostic performance in BC (area under the curve, 0.86; sensitivity = 82%; specificity = 80% at cutoff value 21.4 ng/mL). 8-OHdG is associated with BC risk according to logistic regression analysis. CONCLUSION: We concluded that the significant increase of serum levels of 8-OHdG in patients with BC can be used as a potential noninvasive biomarker for early detection of BC. However, large sample sizes from different stages and types of BC should be included in any future study to confirm the present findings before translating the findings into routine clinical application.

Fallopia japonica, a Natural Modulator, Can Overcome Multidrug Resistance in Cancer Cells.

Resistance of cancer cells to chemotherapy is controlled by the decrease of intracellular drug accumulation, increase of detoxification, and diminished propensity of cancer cells to undergo apoptosis. ATP-binding cassette (ABC) membrane transporters with intracellular metabolic enzymes contribute to the complex and unresolved phenomenon of multidrug resistance (MDR). Natural products as alternative medicine have great potential to discover new MDR inhibitors with diverse modes of action. In this study, we characterized several extracts of traditional Chinese medicine (TCM) plants (N = 16) for their interaction with ABC transporters, cytochrome P3A4 (CYP3A4), and glutathione-S-transferase (GST) activities and their cytotoxic effect on different cancer cell lines. Fallopia japonica (FJ) (Polygonaceae) shows potent inhibitory effect on CYP3A4 P-glycoprotein activity about 1.8-fold when compared to verapamil as positive control. FJ shows significant inhibitory effect (39.81%) compared with the known inhibitor ketoconazole and 100 µg/mL inhibited GST activity to 14 µmol/min/mL. FJ shows moderate cytotoxicity in human Caco-2, HepG-2, and HeLa cell lines; IC50 values were 630.98, 198.80, and 317.37 µg/mL, respectively. LC-ESI-MS were used to identify and quantify the most abundant compounds, emodin, polydatin, and resveratrol, in the most active extract of FJ. Here, we present the prospect of using Fallopia japonica as natural products to modulate the function of ABC drug transporters. We are conducting future study to evaluate the ability of the major active secondary metabolites of Fallopia japonica to modulate MDR and their impact in case of failure of chemotherapy.

Influence of combinations of digitonin with selected phenolics, terpenoids, and alkaloids on the expression and activity of P-glycoprotein in leukaemia and colon cancer cells.

P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, ß-sitosterol-O-glucoside, and ß-carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rho123) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of ß-carotene (40µM) and sanguinarine (1µM) significantly inhibited Rho123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5µM), which also inhibits P-gp, with SM significantly enhanced the inhibition of P-gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p<0.01) and 46% (p<0.01) in Caco-2, and to 61% (p<0.05) and 1% (p<0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells.

Synergism of three-drug combinations of sanguinarine and other plant secondary metabolites with digitonin and doxorubicin in multi-drug resistant cancer cells.

We determined the ability of some phytochemicals, including alkaloids (glaucine, harmine, and sanguinarine), phenolics (EGCG and thymol), and terpenoids (menthol, aromadendrene, ß-sitosterol-O-glucoside, and ß-carotene), alone or in combination with the saponin digitonin to reverse the relative multi-drug resistance of Caco-2 and CEM/ADR5000 cells to the chemotherapeutical agent doxorubicin. The IC(50) of doxorubicin in Caco-2 and CEM/ADR5000 was 4.22 and 44.08µM, respectively. Combination of non-toxic concentrations of individual secondary metabolite with doxorubicin synergistically sensitized Caco-2 and CEM/ADR5000 cells, and significantly enhanced the cytotoxicity of doxorubicin. Furthermore, three-drug combinations (secondary metabolite+digitonin+doxorubicin) were even more powerful. The best synergist was the benzophenanthridine alkaloid sanguinarine. It reduced the IC(50) value of doxorubicin 17.58-fold in two-drug combinations (sanguinarine+doxorubicin) and even 35.17-fold in three-drug combinations (sanguinarine+digitonin+doxorubicin) in Caco-2 cells. Thus synergistic drug combinations offer the possibility to enhance doxorubicin efficacy in chemotherapy.

Digitonin synergistically enhances the cytotoxicity of plant secondary metabolites in cancer cells.

In phytotherapy, extracts from medicinal plants are employed which contain mixtures of secondary metabolites. Their modes of action are complex because the secondary metabolites can react with single or multiple targets. The components in a mixture can exert additive or even synergistic activities. In this study, the cytotoxicity of some phytochemicals, including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, ß-sitosterol-O-glucoside, and ß-carotene) and alkaloids (glaucine, harmine, and sanguinarine) were investigated alone or in combination with the cytotoxic monodesmosidic steroidal saponin digitonin in Caco-2, MCF-7, CEM/ADR5000, and CCRF-CEM cells. Digitonin was combined in non-toxic concentrations (5µM in each cell line; except in MCF-7 the concentration was 2µM), together with a selection of phenolics, terpenoids, and alkaloids to evaluate potential synergistic or additive effects. An enhanced cytotoxicity was observed in most combinations. Even multi-drug resistant (MDR) cells (such as CEM/ADR5000 cells), with a high expression of P-glycoprotein, were responsive to combinations. Sanguinarine was the most cytotoxic alkaloid against CEM/ADR5000, MCF-7, and CCRF-CEM cells alone and in combination with digitonin. As compared to sanguinarine alone, the combination was 44.53-, 15.38-, and 6.65-fold more toxic in each cell line, respectively. Most combinations synergistically increased the cytotoxicity, stressing the importance of synergy when using multi-target drugs and mixtures in phytotherapy.

Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters.

Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (ß-carotene, crocin, retinoic acid, canthaxanthin, and fucoxanthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytometry using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they themselves also function as substrates. Fucoxanthin and canthaxanthin (50-100 µM) produced a 3-5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC(50) values between 100 and 200 µM. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxicity in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respectively as compared to untreated control levels (p<0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy.