Therapeutic benefits of Indole-3-Carbinol in adjuvant-induced arthritis and its protective effect against methotrexate induced-hepatic toxicity.

BACKGROUND: Rheumatoid arthritis (RA) being an incapacitating disease requires early effective intervention. Considering Methotrexate (MTX)- the first line of treatment for RA- intoxicates the liver; therefore, alternative therapies with similar efficacy yet lower cytotoxicity are desired. Indole-3-Carbinol (I3C) which is found in cruciferous vegetables was examined for its possible therapeutic potentials in comparison with MTX by investigating its anti-inflammatory, anti-arthritic, anti-oxidant, and hepatoprotective potentials in adjuvant-induced arthritis (AIA) rat model. METHODS: Arthritis was induced in Sprague Dawley rats by injection of Complete Freund's Adjuvant (CFA). Arthritic rats were treated with I3C and/or MTX. To examine the anti-inflammatory and anti-arthritic effect, the following parameters were assessed: body weight, macroscopic scoring of the hind paw, the level of the pivotal cytokines (TNF-α, IL-6) heavily involved in the pathogenesis, spleen index, and erythrocyte sedimentation rate. At a histological level, the tibiotarsal joint was stained with several specific stains. To assess the hepatoprotective and anti-oxidant effects, several oxidative stress parameters were monitored, and the liver histology was examined. RESULTS: Both I3C and MTX attenuated the inflammation that was aggravated by arthritis by downregulating the inflammatory markers and mediators and alleviating the histopathological changes affecting the tibiotarsal joint. I3C attenuated the liver impairment that was initiated by arthritis and MTX treatment. It did so by downregulating the pro-oxidants and up-regulating the anti-oxidant defenses and by reducing the pathological changes affecting the liver. CONCLUSION: Our results suggest that I3C is as potent as MTX as an anti-inflammatory and anti-arthritic agent. In addition, I3C does so while protecting the liver from damage as opposed to MTX.

Cannabinoid Receptor 2 Gene Polymorphism and the Risk of Developing Rheumatoid Arthritis in Lebanese Patients.

Objective: Evidence supports a role of cannabinoid receptor 2 (CB2) in regulating the immune response. Some variations in the CB2 receptor gene (CB2) were linked to the susceptibility of developing rheumatoid arthritis (RA). The aim of this study is to assess the relationship between CNR2 rs2501431 and the risk of developing RA in Lebanese patients. Methods: A total of one hundred five Lebanese RA patients and one hundred five controls participated in the study. CNR2 was genotyped and analyzed. Results: Using χ2 test, our results show that the CC genotype was the most common (47.6%, p<0.00001) and that the C allele highly predominated (64%, p<0.00001) in the RA group compared to the control group. The relative odds ratio show that carriers of the CC genotype have more than 13-fold risk of developing RA as compared to TT. Conclusion: Our results suggest that the rs2501431 variant of CNR2 gene can be considered as a risk factor for RA development, and thus implicate the potential targeting of CB2 receptor for the treatment of RA.

Silibinin's Effects against Methotrexate-Induced Hepatotoxicity in Adjuvant-Induced Arthritis Rat Model.

Methotrexate (MTX) is the first drug of choice to treat several diseases, including rheumatoid arthritis. However, its administration is accompanied by severe side effects, most commonly hepatotoxicity. Hence, alternative therapies with a lower toxicity and fewer side effects are needed. This study aimed to investigate the antioxidant and hepatoprotective effects of silibinin (SIL, natural agent) against MTX-induced hepatotoxicity in an adjuvant-induced arthritis (AIA) rat model. Arthritic rats were treated with SIL (100 mg/kg) and/or methotrexate (2 mg/kg). Non-arthritic rats, arthritic untreated rats, and arthritic rats who received the vehicle were followed in parallel. SIL alleviated the systemic consequences of arthritis by restoring lost weight, decreasing the erythrocyte sedimentation rate, and ameliorating joint damage, which was evident both micro- and macroscopically. Additionally, SIL prevented the histopathological alterations in the liver and significantly reduced the liver damage caused by MTX and AIA, as shown by a decrease in the markers of liver damage (ALT and AST). Furthermore, SIL relieved the oxidative stress induced by AIA and MTX in liver tissue by decreasing the lipid peroxidation (MDA) levels and enhancing the antioxidant defense system (GSH levels; catalase and superoxide dismutase (SOD) activities). In conclusion, our results suggest that SIL is a potent antioxidant and hepatoprotective agent in arthritic rats. It markedly attenuated the progression and severity of the arthritic disease and eased the oxidative stress in liver tissue by improving the pro-oxidant/antioxidant balance.

Rosemary essential oil potentiates the antitumour activity of 5-fluorouracil in human colorectal carcinoma cells.

OBJECTIVES: Improving response rates in colorectal cancer (CRC) is an urgent clinical need. This study aimed to explore the synergistic action of Lebanese rosemary essential oil (REO) and 5-fluorouracil (5-FU) in HCT116 CRC cells. METHODS: We tested the cell viability of monotherapy and combination therapy. The combination index was calculated using CompuSyn software to evaluate drug-drug interactions and the level of synergistic cytotoxicity. We also evaluated cell migration and cytopathology. Furthermore, cell apoptosis-related proteins (i.e. Bax and Bcl-2) were measured by Western blot analysis. KEY FINDINGS: The REO/5-FU combination synergistically reduced cell viability, effectively decreased cell migration, and increased the Bax/Bcl-2 ratio in HCT116 cells. This triggered a proapoptotic morphology and initiated an apoptotic cascade in HCT116 cells, as indicated by a higher Bax/Bcl-2 ratio. CONCLUSIONS: Our results provide evidence of the REO/5-FU combination as a better approach to improve 5-FU anticancer efficacy and allow the use of lower 5-FU doses due to the adjuvant effect of REO.

Molecular properties prediction, anticancer and anti-inflammatory activities of some pyrimido[1,2-b]pyridazin-2-one derivatives.

Introduction: The anticancer and anti-inflammatory activities of a novel series of eleven pyrimido[1,2-b]pyridazin-2-one analogues substituted at position 7 were assessed in the current study. Methods: The physicochemical characteristics were studied using MolSoft software. The antiproliferative activity was investigated by MTT cell viability assay, and cell cycle analysis elucidated the antiproliferative mechanism of action. Western blot analysis examined the expression levels of key pro-apoptotic (Bax, p53) and pro-survival (Bcl-2) proteins. The anti-inflammatory activity was assessed by measuring the production levels of nitric oxide in RAW264.7 cells, and the expression levels of COX-2 enzyme in LPS-activated THP-1 cells. In addition, the gene expression of various pro-inflammatory cytokines (IL-6, IL-8, IL-1ß, TNF-α) and chemokines (CCL2, CXCL1, CXCL2, CXCL3) was assessed by RT-qPCR. Results: Compound 1 bearing a chlorine substituent displayed the highest cytotoxic activity against HCT-116 and MCF-7 cancer cells where IC50 values of 49.35 ± 2.685 and 69.32 ± 3.186 µM, respectively, were achieved. Compound 1 increased the expression of pro-apoptotic proteins p53 and Bax while reducing the expression of pro-survival protein Bcl-2. Cell cycle analysis revealed that compound 1 arrested cell cycle at the G0/G1 phase. Anti-inflammatory assessments revealed that compound 1 displayed the strongest inhibitory activity on NO production with IC50 of 29.94 ± 2.24 µM, and down-regulated the expression of COX-2. Compound 1 also induced a statistically significant decrease in the gene expression of various cytokines and chemokines. Conclusion: These findings showed that the pyrimidine derivative 1 displayed potent anti-inflammatory and anticancer properties in vitro, and can be selected as a lead compound for further investigation.

Proapoptotic and Antiproliferative Effects of the Desert Truffle Terfezia boudieri on Colon Cancer Cell Lines.

Background: Colon cancer is the second leading cause of cancer-related mortality, and ranks third among cancers in terms of prevalence. Despite advances in early detection and treatment with chemotherapy and surgery, colon cancer continues to be associated with high recurrence rates, thereby resulting in a heavy disease burden. Moreover, the effectiveness of currently available treatment modalities is limited by the occurrence of toxic side effects. Hence, there is an urgent need to develop alternative treatments. Extracts from the black desert truffle Terfezia boudieri (T. boudieri) have shown promising anticancer properties. However, the cellular mechanisms underlying this activity remain poorly understood. Methods: In this study, the colon cancer cell lines HCT-116 and Caco-2 were treated with either water or ethanolic extract of T. boudieri. Cell viability and the half-maximal inhibitory concentration were determined using MTT assays. Then, the activity of the more potent water extract was further verified using crystal violet assays, and its role in inhibiting colony formation and wound healing was investigated. Protein levels of p53, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), cyclin D1 (CCND1), and c-Myc were measured in cells treated with different doses of the water extract. Results: Treatment with the water extract of T. boudieri reduced the capacity of cells for wound healing and colony formation in a dose-dependent manner. The Bax/Bcl-2 ratio and p53 expression were elevated in both cell lines. In contrast, the levels of cyclin D1 and c-Myc were suppressed. Conclusion: T. boudieri water extract exerted a cytotoxic effect on colon cancer cells, and blocked colony formation and wound healing potentially through inhibition of proliferation. Mechanistically, these effects are attributed to influence the mitochondrial pathway of apoptosis, proteins involved in cellular proliferation, and the cell cycle.

Synergistic Effect of Epigenetic Inhibitors Decitabine and Suberoylanilide Hydroxamic Acid on Colorectal Cancer In vitro.

BACKGROUND: Colorectal Cancer (CRC) is a common cause of oncological deaths worldwide. Alterations of the epigenetic landscape constitute a well-documented hallmark of CRC phenotype. The accumulation of aberrant DNA methylation and histone acetylation plays a major role in altering gene activity and driving tumor onset, progression and metastasis. OBJECTIVE: In this study, we evaluated the effect of Suberoylanilide Hydroxamic Acid (SAHA), a panhistone deacetylase inhibitor, and Decitabine (DAC), a DNA methyltransferase inhibitor, either alone or in combination, on Caco-2 human colon cancer cell line in vitro. RESULTS: Our results showed that SAHA and DAC, separately, significantly decreased cell proliferation, induced apoptosis and cell cycle arrest of Caco-2 cell line. On the other hand, the sequential treatment of Caco-2 cells, first with DAC and then with SAHA, induced a synergistic anti-tumor effect with a significant enhancement of growth inhibition and apoptosis induction in Caco-2 cell line as compared to cells treated with either drug alone. Furthermore, the combination therapy upregulates protein expression levels of pro-apoptotic proteins Bax, p53 and cytochrome c, downregulates the expression of antiapoptotic Bcl-2 protein and increases the cleavage of procaspases 8 and 9; this suggests that the combination activates apoptosis via both the intrinsic and extrinsic pathways. Mechanistically, we demonstrated that the synergistic anti-neoplastic activity of combined SAHA and DAC involves an effect on PI3K/AKT and Wnt/ß-catenin signaling. CONCLUSION: In conclusion, our results provide evidence for the profound anti-tumorigenic effect of sequentially combined SAHA and DAC in the CRC cell line and offer new insights into the corresponding underlined molecular mechanism.

Adjuvant Epigenetic Therapy of Decitabine and Suberoylanilide Hydroxamic Acid Exerts Anti-Neoplastic Effects in Acute Myeloid Leukemia Cells.

Atypical epigenetic processes including histone acetylation and DNA methylation have been identified as a fundamental theme in hematologic malignancies. Such mechanisms modify gene expression and prompt, in part at least, the initiation and progression of several malignancies including acute myeloid leukemia. In the current study we determined the effects of treating KG-1 and U937 acute myeloid leukemia (AML) cells, in vitro, with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), or with a DNMT inhibitor, decitabine (DAC), or their combination, on cell proliferation, cell cycle progression, apoptosis, and expression of apoptosis-related proteins. Each of SAHA and DAC attenuated cell proliferation and induced cell cycle arrest and apoptotic cell death of KG-1 and U937 cell lines. Besides, their sequential combination improved the obtained anti-neoplastic effect: significant augmentation of growth inhibition and apoptosis induction as compared to cells treated with either drug alone. This effect was featured by the upregulated expression of Bax, cytochrome c1, p21, and cleaved caspases 8, 9, and 3, signifying the activation of both the intrinsic and extrinsic pathways of apoptosis. The sequential combination of SAHA and DAC causes a profound antitumorigenic effect in AML cell lines by inducing the expression of tumor suppressor genes.

Study of cannabinoid receptor 2 Q63R gene polymorphism in Lebanese patients with rheumatoid arthritis.

The cannabinoid (CB) receptor 2, primarily expressed in immune cells, was shown to play important immune-regulatory functions. In particular, the CB2-R63 functional variant has been shown to alter the ability of the CB2 receptor to exert its inhibitory function on T lymphocytes. The aim of this study was to investigate the association between a common dinucleotide polymorphism, Q63R, in the cannabinoid receptor 2 gene (CNR2) and rheumatoid arthritis (RA) in the Lebanese population. One hundred five unrelated Lebanese RA patients and one hundred five controls from different Lebanese governorates were recruited in this study. Genomic DNA was extracted, polymerase chain reaction was performed, and CNR2 was genotyped in a blinded fashion. The χ2 test was used to determine the differences in genotypes and allele frequencies. CNR2 genotyping showed significantly higher frequencies of the CB2-R63 variant (allele frequencies, P < 0.00001; genotype distribution, P < 0.00001) in RA patients when compared with healthy controls. Moreover, RR carriers had more than 10-fold risk for developing RA (OR = 10.8444, 95% CI = 5.0950-23.0818; P < 0.0001), and QR carriers had more than 3-fold risk (OR = 3.8667, 95% CI = 1.7886-8.3591; P = 0.0006) as compared with QQ carriers. Our preliminary results suggest a role of CB2-Q63R gene polymorphism in the etiology of RA, thus supporting its potential use as a pharmacological target for selective agonists in clinical practice.

Anti-Inflammatory, Antioxidative, and Hepatoprotective Effects of Trans Δ9-Tetrahydrocannabinol/Sesame Oil on Adjuvant-Induced Arthritis in Rats.

Rheumatoid arthritis (RA) is a painful chronic autoimmune disease affecting the joints. Its first-line therapy, Methotrexate (MTX), although effective in ameliorating the progress of the disease, induces hepatotoxicity over long-term usage. Thus, seeking natural compounds with fewer side effects could be an alternative therapeutic approach. This study aimed to investigate the anti-inflammatory, antiarthritic, and antioxidative effects of synthetic trans-Δ9-tetrahydrocannabinol (Δ9-THC) dissolved in sesame oil (Dronabinol) against MTX in adjuvant-induced arthritis (AIA) rat model. Daily oral administration of Δ9-THC/sesame oil, over a period of 21 days, was well tolerated in arthritic rats with no particular psychoactive side effects. It markedly attenuated the severity of clinical manifestations, recovered the histopathological changes in tibiotarsal joints, and repressed the splenomegaly in arthritic rats. Δ9-THC/sesame oil therapy showed similar effects to MTX in neutralizing the inflammatory process of AIA, through attenuating erythrocyte sedimentation rate (ESR) scores and proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1ß), and interleukin-6 (IL-6) levels, to normal values. As opposed to MTX, this natural combination markedly protected the liver of arthritic rats and downregulated the induced oxidative stress by increasing the antioxidant defense system such as activities of catalase and superoxide dismutase (SOD) and levels of glutathione (GSH). These results suggest promising effects for the clinical use of Δ9-THC/sesame oil therapy in alleviating arthritic clinical signs as well as arthritis-induced liver injury.

Enhanced magnitude and breadth of neutralizing humoral response to a DNA vaccine targeting the DHBV envelope protein delivered by in vivo electroporation.

We explored in the duck hepatitis B virus (DHBV) model the impact of electroporation (EP)-mediated DNA vaccine delivery on the neutralizing humoral response to viral preS/S large envelope protein. EP enhanced the kinetics and magnitude of anti-preS response compared to the standard needle DNA injection (SI). Importantly, EP dramatically enhanced the neutralizing potency of the humoral response, since antibodies induced by low DNA dose (10 µg) were able to highly neutralize DHBV and to recognize ten antigenic regions, including four neutralization epitopes. Whereas, SI-induced antibodies by the same low DNA dose were not neutralizing and the epitope pattern was extremely narrow, since it was limited to only one epitope. Thus, EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose.

In vivo electroporation improves therapeutic potency of a DNA vaccine targeting hepadnaviral proteins.

This preclinical study investigated the therapeutic efficacy of electroporation (EP)-based delivery of plasmid DNA (pDNA) encoding viral proteins (envelope, core) and IFN-γ in the duck model of chronic hepatitis B virus (DHBV) infection. Importantly, only DNA EP-therapy resulted in a significant decrease in mean viremia titers and in intrahepatic covalently closed circular DNA (cccDNA) levels in chronic DHBV-carrier animals, compared with standard needle pDNA injection (SI). In addition, DNA EP-therapy stimulated in all virus-carriers a humoral response to DHBV preS protein, recognizing a broader range of major antigenic regions, including neutralizing epitopes, compared with SI. DNA EP-therapy led also to significant higher intrahepatic IFN-γ RNA levels in DHBV-carriers compared to other groups, in the absence of adverse effects. We provide the first evidence on DNA EP-therapy benefit in terms of hepadnaviral infection clearance and break of immune tolerance in virus-carriers, supporting its clinical application for chronic hepatitis B.