Anti-Cancer Effect of Bromelain and Its Combination with Cisplatin on HN5 Cell Line (Squamous Cell Carcinoma).

Statement of the Problem: Squamous cell carcinoma (SCC) comprises over 90% of oral malignancies. Cisplatin, as a selective chemotherapy agent to treat SCC, has many side effects despite its high effectiveness. There are some studies on the effects of bromelain derived from pineapple stems on different malignancies. Purpose: The aim of this study was to investigate the effect of bromelain alone and in combination with Cisplatin on oral squamous cell carcinoma (OSCC) and fibroblast cell lines. Materials and Method: In this interventional study, the HN5 cell line of OSCC and fibroblast cell line were treated with different concentrations of bromelain alone and in combination with cisplatin. Cell viability test was performed after 24, 48 and 72 hours using MTT (3-)4,5-dimethylthiazol-2-yl(-2,5 diphenyl tetrazolium bromide) assay. In the final stage, the drug-treated cells underwent flow cytometry to assess apoptosis patterns. Data were analyzed using SPSS 17, ANOVA (for general comparison of groups) and LSD post hoc tests (for comparison two groups). p< 0.05 was considered statistically significant. Results: The findings suggested that although bromelain showed toxic effects on HN5 cancer cells, its combination with Cisplatin resulted in little improvement in its effectiveness. Bromelain alone and in combination with Cisplatin presented cytotoxic effects against fibroblasts, which depended on the dosage and time exposure (p< 0.05). The flow cytometry results did not support the superior effect of the combination of two medications over Cisplatin alone (p> 0.05). Conclusion: According to the findings, although adding bromelain to Cisplatin reduced toxicity on normal tissues, the combination of these two drugs did not increase the anticancer effect of Cisplatin. Thus, bromelain in combination with Cisplatin is not recommended as an adjuvant drug for OSCC.

In silico design of a TLR4-mediating multiepitope chimeric vaccine against amyotrophic lateral sclerosis via advanced immunoinformatics.

Amyotrophic lateral sclerosis (ALS) is the most prevalent motor neuron disorder worldwide. In ALS, progressing disease can result from misfolding and aggregation of superoxide dismutase-1 (SOD1) or TAR DNA-binding protein 43 kDa (TDP43). An efficient immunotherapy for ALS should spare intact SOD1 while eliminating its dysfunctional variant. We utilized advanced immunoinformatics to suggest a potential vaccine candidate against ALS by proposing a model of dynamic TLR4 mediation and induction of a specific Th2-biased shift against mutant SOD1, TDP43, and TRAF6, a protein that specifically interacts with dysfunctional SOD1. SOD1, TDP43, and TRAF6 were retrieved in FASTA. Immune Epitopes Database and CTLpred suggested T/B-cell epitopes from disease-specific regions of selected antigens. A TLR4-mediating adjuvant, RS01, was used. Sequences were assembled via suitable linkers. Tertiary structure of the protein was calculated. Refined protein structure and physicochemical features of the 3D structure were verified in silico. Differential immune induction was assessed via C-ImmSim. GROningen MAchine for Chemical Simulation was used to assess evolution of the docked vaccine-TLR4 complex in blood. Our protein showed high structural quality and was nonallergenic and immune inducing. Also, the vaccine-TLR4 complex stability was verified by RMSD, RMSF, gyration, and visual analyses of the molecular dynamic trajectory. Contact residues in the vaccine-TLR4 complex showed favorable binding energies. Immune stimulation analyses of the proposed candidate demonstrated a sustained memory cell response and a strong adaptive immune reaction. We proposed a potential vaccine candidate against ALS and verified its physicochemical and immune inducing features. Future studies should assess this vaccine in animal studies.

Decrease of intracellular ROS by arbutin is associated with apoptosis induction and downregulation of IL-1ß and TNF-α in LNCaP; prostate cancer.

Increased reactive oxygen species (ROS) along with inflammation are involved in the prostate cancer (PCa). Therefore, this study was conducted to investigate the molecular mechanisms that were affected by arbutin as an antioxidant on prostate cancer cell line; LNCap. The intracellular ROS measurement confirmed that arbutin significantly (p < .05) decreased the ROS levels in a dose-dependent manner. Detection of cell death profile established that 1,000 µM of arbutin could remarkably induced apoptosis (p < .05), while tert-butyl hydroperoxide (tBHP) as ROS inducer prompted necrosis. In addition, 1,000 µM of arbutin successfully decreased expressions of IL-1ß and TNF-α genes (p < .05). Furthermore, evaluation of the IL-1ß protein level showed that arbutin could significantly decrease this cytokine (p < .05). In summary, reduction of ROS along with increasing apoptosis and decreasing expression of pro-inflammatory genes following arbutin treatment can open new visions in the treatment of prostate cancer using complementary medicine. PRACTICAL APPLICATIONS: Nowadays, arbutin as a glycosylated hydroquinone is available commercially in both natural and synthetic forms. Arbutin is of interest because of its skin-lightening effect, and used in cosmetic products for cutaneous hyperpigmentation. Arbutin inhibited tyrosinase in melanocytes competitively. Moreover, arbutin was able to attenuate oxidative stress and, its anti-inflammatory activities has been established. In addition, arbutin has represented useful activities for suppression of malignant melanoma development. In addition, arbutin exhibits several pharmacological effects, including antimicrobial, antihyperlipidemic, antihyperglycemic, and alpha amylase inhibitory effects. In this study, we showed its effect on prostate cancer in vitro. Therefore, it opens new insights in the complementary medicine that can maintain or improve human health.

Iranian propolis efficiently inhibits growth of oral streptococci and cancer cell lines.

BACKGROUND: Propolis is a natural bee product with a wide range of biological activities that are related to its chemical composition. The present study investigated the quantification of quercetin (Q) in Ardabil ethanol extract of propolis (AEEP), and then compared its anti-bacterial, anti- biofilm and cytotoxic effects on cancer and normal cell lines. METHOD: In the present study, the chemical composition of AEEP was determined through the high-performance liquid chromatography (HPLC). The AEEP and its main component, quercetin (Q), were evaluated in vitro against 57 oral streptococci by a broth micro-dilution method. The biofilm formation was assessed through the crystal violet staining and MTT assays. The impact of AEEP and Q anti-proliferative effect were evaluated on the fibroblast as normal and cancer cell lines (KB and A431). RESULTS: The Q concentration in the composition of AEEP was 6.9% of all its components. The findings indicated that the AEEP and Q were efficient against the cariogenic bacteria and were able to inhibit the S.mutans biofilm adherence at a sub-MIC concentration. Moreover, electron micrographs indicated the inhibition of biofilms compared to control biofilms. In addition, the AEEP and Q indicated a dose-dependent cytotoxic effect on A431 and KB cell lines. On the contrary, they had no cytotoxic effect on fibroblast cells. CONCLUSION: The results indicated that the synergistic impact of main components of AEEP was related to the inhibition of the cancer cell proliferation, cariogenic bacteria and oral biofilm formation. It may play a promising role in the complementary medicine and, it is suggested to be used as food additives.

Propolis attenuates lipopolysaccharide-induced inflammatory responses through intracellular ROS and NO levels along with downregulation of IL-1ß and IL-6 expressions in murine RAW 264.7 macrophages.

Propolis had a wide spectrum of biological activities. In the current study, antioxidative and the immunomodulatory effects of the Polur ethanol extract of propolis (PEEP) in murine macrophage (RAW 264.7) cells were investigated. Bioactive composition of the PEEP was determined by HPLC analysis. Cells were treated with different concentrations of PEEP and LPS, then cell viability, NO levels, and expression of inflammatory factors were evaluated. HPLC analysis of PEEP indicated the presence of flavonoids and phenolic acid. The PEEP inhibited the proliferation of RAW 264.7 cells with IC50 15 ± 3.2 µg/ml. Reactive oxygen species (ROS) and NO production was significantly reduced by 0.15 µg/ml of PEEP. Additionally, expression of Cox-2, IL-1ß and IL-6 significantly decreased. The obtained results supported the PEEP anti-inflammatory effects on RAW 264.7 cells may be applied via reducing ROS and NO production along with COX-2, IL-1ß, and IL-6 expression. PRACTICAL APPLICATIONS: Propolis is a resinous substance produced by the honeybee that has been adopted as a form of traditional medicine since ancient times. The main compounds found in propolis are typically various and depend on the type of plants and climatic region. In this respect, a wide spectrum of biological activities for propolis has been identified including antioxidant, antimicrobial, anticarcinogenic, anti-inflammatory, as well as antifungal properties. This extraordinary substance is rich in flavonoids and antioxidants. Therefore, it is now widely used in foods and drinks with the claim that it can maintain or improve human health.

Immunomodulatory effects of Thymol through modulation of redox status and trace element content in experimental model of asthma.

Oxidative stress plays a key role in the immunopathogenesis of asthma. The objective of this study was to investigate the thymol effects on oxidative parameters along with trace elements in asthma experimental model. The Balb/c mice were sensitized by intraperitoneal injection of ovalbumin and thymol (8, 16 and 32 mg/kg) and dexamethasone (DEX) (2 mg/kg) were orally administered to sensitized mice. Oxidative stress parameters including protein carbonyl content, malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and total antioxidant capacity (TAC) besides trace element levels were evaluated. The protein carbonyl content, MDA and 8-OHdG in treated mice with 32 mg/kg of thymol significantly decreased compared to asthmatic mice (P < 0.01). Also, TAC significantly increased (P < 0.001) as well as zinc and selenium levels while copper level decreased. 16 mg/kg of thymol reduced the protein carbonyl content, MDA and 8-OHdG compared to asthmatic mice (P < 0.05). In addition, thymol improved the most prominent inflammation characteristics of asthma. The obtained results suggest that thymol has a protective effect against oxidative stress and it was also able to partially restore the defective trace element levels in asthma. Based on our observations, thymol may be used for alternative / complementary therapy in asthma.

In silico analyses of heat shock protein 60 and calreticulin to designing a novel vaccine shifting immune response toward T helper 2 in atherosclerosis.

Recent experiments demonstrated that atherosclerosis is a Th1 dominant autoimmune condition, whereas Th2 cells are rarely detected within the atherosclerotic lesions. Several studies have indicated that Th2 type cytokines could be effective in the reduction and stabilization of atherosclerotic plaque. Therefore, the modulation of the adaptive immune response by shifting immune responses toward Th2 cells by a novel vaccine could represent a promising approach to prevent from progression and thromboembolic events in coronary artery disease. In the present study, an in silico approach was applied to design a novel multi-epitope vaccine to elicit a desirable immune response against atherosclerosis. Six novel IL-4 inducing epitopes were selected from HSP60 and calreticulin proteins. To enhance epitope presentation, IL-4 inducing epitopes were linked together by AAY and HEYGAEALERAG linkers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Moreover, cholera toxin B (CTB) was employed as an adjuvant. A multi-epitope construct was designed based on predicted epitopes which was 320 residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this chimeric protein were analyzed using bioinformatics tools and servers. Based on bioinformatics analysis, a soluble, and non-allergic protein with 35.405kDa molecular weight was designed. Expasy ProtParam classified this chimeric protein as a stable protein. In addition, predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell mediated immune response and shift immune responses toward protective Th2 immune response. Various in silico analyses indicate that this vaccine is a qualified candidate for improvement of atherosclerosis by inducing immune responses toward T helper 2.

Immunotherapy with a recombinant hybrid molecule alleviates allergic responses more efficiently than an allergenic cocktail or pollen extract in a model of chenopodium album allergy.

BACKGROUND: The aim of this study is to assess the therapeutic potential of a recombinant hybrid molecule (rHM) alongside an allergenic cocktail from recombinant wild-type allergens as well as pollen extract on Chenopodium album allergy, using a BALB/c mouse model. METHODS: The BALB/c mice had already been sensitized to C. album via intraperitoneal injections of alum-adsorbed allergenic cocktail and immunotherapy procedure was followed by subcutaneous injections of the rHM, allergenic cocktail and pollen extract at weekly intervals. Humoral immune responses were determined via measurement of specific antibodies in serum. Splenocytes of immunized mice were stimulated in vitro and then proliferation responses, cytokine secretion and mRNA expression of genes involved in immunotherapy were examined by ELISA and real-time PCR. RESULTS: Sensitized mice were identified with high specific IgE against allergenic cocktail when compared with healthy mice. Immunotherapy with the rHM induced the highest ratio of the IgG2a/IgG1 levels compared to allergenic cocktail or C. album pollen extract. The rHM was able to induce proliferative responses as well as the allergenic cocktail in cultured splenocytes. Immunotherapy with the rHM significantly improved secretion of IFN-γ and IL-10, while secretion of IL-13 rapidly diminished. Interestingly, mRNA expression of GATA3 was strongly decreased in rHM-treated mice whereas mRNA expression of T-bet and Foxp3 was significantly increased. CONCLUSION: Our results prove that immunotherapy with the rHM effectively controlled allergic responses by shifting from a Th2-like immune response to a Th1-dominated immune response.

Constructing a hybrid molecule with low capacity of IgE binding from Chenopodium album pollen allergens.

Allergen specific immunotherapy is the only remedy to prevent the progression of allergic diseases. Nowadays, using of recombinant allergens with reduced IgE-binding capacity is an ideal tool for allergen immunotherapy. Therefore, in this study we focused on a hybrid molecule (HM) production with reduced IgE reactivity from Chenopodium album pollen allergens. By means of genetic engineering, a head to tail structure of the three allergens of the C. album pollen was designed. The resulting DNA construct coding for a 46kDa HM was inserted into an expression vector and expressed as hexahistidine tagged fusion protein in Escherichia coli. IgE reactivity of the HM was evaluated by western blotting, inhibition ELISA and in vivo skin prick test and its immunogenic property was tested by proliferation assay. The recombinant HM was expressed and purified by nickel-affinity chromatography. Comparison of the recombinant HM with a mixture of three recombinant allergens, as well as natural allergens in the whole C. album pollen extract via immunological experiments revealed that it has a much lower potential of IgE reactivity. Furthermore, in vivo skin prick tests showed that it has a significantly lower potency to induce cutaneous reactions than the mixture of recombinant wild type allergens and whole extract while, it had been preserved immunogenic properties. Our results have demonstrated that assembling three allergens of C. album in a hybrid molecule can reduce its IgE reactivity.

Diagnosis of Chenopodium album allergy with a cocktail of recombinant allergens as a tool for component-resolved diagnosis.

Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens, so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail. The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could be replaced with natural C. album allergen extracts in skin prick test and serologic tests.