A High-throughput Quantitative Expression Analysis of Cancer-related Genes in Human HepG2 Cells in Response to Limonene, a Potential Anticancer Agent.

BACKGROUND: Citrus bioactive compounds, as active anticancer agents, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. OBJECTIVES: The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. METHODS: The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of the pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. Highthroughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. RESULTS: In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene- driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from the most to the least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. CONCLUSION: The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines.

The potential of bacteriophage cocktail in eliminating Methicillin-resistant Staphylococcus aureus biofilms in terms of different extracellular matrices expressed by PIA, ciaA-D and FnBPA genes.

BACKGROUND: This study assessed novel approach of using highly lytic phages against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) biofilms with and without biofilm extracellular matrix- disrupting chemical. METHOD: The resultant phage-based control was assessed in relation to the type of biofilm extracellular matrix namely, polysaccharide intercellular adhesion (PIA) or proteinacious fibronectin-binding protein A (FnBPA). The biofilms were formed in vitro by 24 h incubation of bacteria in 96 wells microtiter plates at room temperature. The formed biofilms were assessed by tissue culture plate (TCP). Moreover, the nature of the biofilm was assessed by scanning electron microscopy (SEM) and PCR assay for detecting PIA genes, ciaA-D and FnBPA genes. RESULTS: this study showed that applied phages with 0.08 % benezenthonium chloride, for PIA biofilms, and 0.06 % ethanol, for proteinacious FnBPA biofilms, exerted 100 % eradication for MSSA biofilms and about 78 % of MRSA biofilms. The phage-based control of biofilms with chemical adjuvant showed significantly higher efficiency than that without adjuvant (P < 0.05). Moreover, FnBPA biofilms were more common in MRSA than in MSSA while PIA biofilms were more common in MSSA than in MRSA. And the most resistant type of biofilms to phage-based control was FnBPA in MRSA where 50 % of biofilms were reduced but not eradicated completely. CONCLUSIONS: It is concluded that PIA-disturbing agent and protein denaturing alcohol can increase the efficiency of attacking phages in accessing host cell walls and lysing them which in turn lead to much more efficient MRSA and MSSA biofilm treatment and prevention.

Novel antiviral activity of mung bean sprouts against respiratory syncytial virus and herpes simplex virus -1: an in vitro study on virally infected Vero and MRC-5 cell lines.

BACKGROUND: New sources for discovering novel antiviral agents are desperately needed. The current antiviral products are both expensive and not very effective. METHODS: The antiviral activity of methanol extract of mung bean sprouts (MBS), compared to Ribavarin and Acyclovir, on respiratory syncytial virus (RSV) and Herpes Simplex virus -1 (HSV-1) was investigated using cytotoxicity, virus yield reduction, virucidal activity, and prophylactic activity assays on Vero and MRC-5 cell lines. Moreover, the level of antiviral cytokines, IFNß, TNFα, IL-1, and IL-6 was assessed in MBS-treated, virally infected, virally infected MBS-treated, and control groups of MRC-5 cells using ELISA. RESULTS: MBS extract showed reduction factors (RF) 2.2 × 10 and 0.5 × 10(2) for RSV and HSV-1, respectively. The 2 h incubation virucidal and prophylactic selectivity indices (SI) of MBS on RSV were 14.18 and 12.82 versus Ribavarin SI of 23.39 and 21.95, respectively, and on HSV-1, SI were 18.23 and 10.9 versus Acyclovir, 22.56 and 15.04, respectively. All SI values were >10 indicating that MBS has a good direct antiviral and prophylactic activities on both RSV and HSV-1. Moreover, interestingly, MBS extract induced vigorously IFNß, TNFα, IL-1, and IL-6 cytokines in MRC-5 infected-treated group far more than other groups (P < 0.05) and induced TNFα and IL-6 in treated group more than infected group (P < 0.05). CONCLUSIONS: MBS extract has potent antiviral and to a lesser extent, prophylactic activities against both RSV and HSV-1, and in case of HSV-1, these activities were comparable to Acyclovir. Part of the underlying mechanism(s) of these activities is attributed to MBS potential to remarkably induce antiviral cytokines in human cells. Hence, we infer that MBS methanol extract could be used as such or as purified active component in protecting and treating RSV and HSV-1 infections. More studies are needed to pinpoint the exact active components responsible for the MBS antiviral activities.

Novel molecular, cytotoxical, and immunological study on promising and selective anticancer activity of mung bean sprouts.

BACKGROUND: The anticancer and immunomodulatory activity of mung bean sprouts (MBS) and the underlying mechanisms against human cervical and hepatocarcinoma cancer cells were explored. METHODS: MBS cytotoxicity and MBS-induced anticancer cytokines, TNF-α and IFN-ß from cancer cells, and immunological cytokines, IL-4, IFN-γ, and IL-10 from peripheral mononuclear cells (PMNC) were assessed by MTS and ELISA assays. Apoptotic cells were investigated by flow cytometry. The expression level of apoptotic genes (Bax, BCL-2, Capsases 7-9) and cell cycle regulatory genes (cyclin D, E, and A) and tumor suppressor proteins (p27, p21, and p53) was assessed by real-time qPCR in the cancer cells treated with extract IC50. RESULTS: The cytotoxicity on normal human cells was significantly different from HeLa and HepG2 cells, 163.97 ± 5.73, 13.3 ± 0.89, and 14.04 ± 1.5 mg/ml, respectively. The selectivity index (SI) was 12.44 ± 0.83 for HeLa and 11.94 ± 1.2 for HepG2 cells. Increased levels of TNF-α and IFN-ß were observed in the treated HeLa and HepG2 culture supernatants when compared with untreated cells. MBS extract was shown to be an immunopolarizing agent by inducing IFNγ and inhibiting IL-4 production by PBMC; this leads to triggering of CMI and cellular cytotoxicity. The extract induced apoptosis, in a dose and time dependent manner, in treated HeLa and HepG2, but not in untreated, cells (P < 0.05). The treatment significantly induced cell cycle arrest in G0/G1 in HeLa cells. The percentage of cells in G0/G1 phase of the treated HeLa cells increased from 62.87 ± 2.1%, in untreated cells, to 80.48 ± 2.97%. Interestingly, MBS IC50 induced the expression of apoptosis and tumor suppressor related genes in both HeLa and HepG2 cells. MBS extract succeeded in inducing cdk-inhibitors, p21, p53, and p27 in HeLa cells while it induced only p53 in HepG2 cells (P < 0.05). This is a clue for the cell type- specific interaction of the studied extract. These proteins inhibit the cyclin-cdk complexes apart from the presence of some other components that might stimulate some cyclins such as cyclin E, A, and D. CONCLUSION: MBS extract was shown to be a potent anticancer agent granting new prospects of anticancer therapy using natural products.

Inhibition of growth of highly resistant bacterial and fungal pathogens by a natural product.

The continuous escalation of resistant bacteria against a wide range of antibiotics necessitates discovering novel unconventional sources of antibiotics. B. oleracea L (red cabbage) is health-promoting food with proven anticancer and anti-inflammatory activities. However, it has not been researched adequately for its antimicrobial activity on potential resistant pathogens. The methanol crude extract of B. oleracea L. was investigated for a possible anti-microbial activity. The screening method was conducted using disc diffusion assay against 22 pathogenic bacteria and fungi. It was followed by evaluation of the minimum inhibitory concentration (MIC). Moreover, the antibacterial and the antifungal activities were confirmed using the minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC), respectively. Remarkable, antibacterial activity was evident particularly against highly infectious microorganisms such as Methicillin-resistant Staphylococcus aureus, Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Salmonella enterica serovar Typhimurium as well as against human fungal pathogens, Trichophyton rubrum and Aspergillus terreus. Red cabbage is a rich source of phenolic compounds, anthocyanins being the most abundant class, which might explain its potent antimicrobial action. This extract is potentially novel for future antimicrobials, inexpensive, and readily available at a large scale for pharmaceutical companies for further investigation and processing.

The association of Streptococcus bovis/gallolyticus with colorectal tumors: the nature and the underlying mechanisms of its etiological role.

Streptococcus bovis (S. bovis) bacteria are associated with colorectal cancer and adenoma. S. bovis is currently named S. gallolyticus. 25 to 80% of patients with S. bovis/gallolyticus bacteremia have concomitant colorectal tumors. Colonic neoplasia may arise years after the presentation of bacteremia or infectious endocarditis of S. bovis/gallolyticus. The presence of S. bovis/gallolyticus bacteremia and/or endocarditis is also related to the presence of villous or tubular-villous adenomas in the large intestine. In addition, serological relationship of S. gallolyticus with colorectal tumors and direct colonization of S. gallolyticus in tissues of colorectal tumors were found. However, this association is still under controversy and has long been underestimated. Moreover, the etiological versus non-etiological nature of this associationis not settled yet. Therefore, by covering the most of up to date studies, this review attempts to clarify the nature and the core of S. bovis/gallolyicus association with colorectal tumors and analyze the possible underlying mechanisms.

Molecular detection, quantification, and isolation of Streptococcus gallolyticus bacteria colonizing colorectal tumors: inflammation-driven potential of carcinogenesis via IL-1, COX-2, and IL-8.

BACKGROUND: Colorectal cancer (CRC) has long been associated with bacteremia and/or endocarditis by Streptococcus gallolyticus member bacteria (SGMB) but the direct colonization of SGMB along with its molecular carcinogenic role, if any, has not been investigated. We assessed the colonization of SGMB in CRC patients with history of bacteremia (CRC-w/bac) and without history of bacteremia (CRC-wo/bac) by isolating SGMB from feces, mucosal surfaces of colorectum, and colorectal tissues and detecting SGMB DNA, via PCR and in situ hybridization (ISH) assays targeting SodA gene in colorectal tissues. Moreover, mRNA of IL1, IL-8, COX-2, IFN-γ, c-Myc, and Bcl-2 in colorectal tissues of studied groups was assessed via ISH and RT-PCR. RESULTS: SGMB were found to be remarkably isolated in tumorous (TU) and non-tumorous (NTU) tissues of CRC-w/bac, 20.5% and 17.3%, and CRC-wo/bac, 12.8% and 11.5%, respectively while only 2% of control tissues revealed SGMB (P < 0.05); such contrast was not found in mucosal and fecal isolation of SGMB. The positive detection of SGMB DNA in TU and NTU of CRC-w/bac and CRC-wo/bac via PCR, 48.7%, 35.9%, 32.7%, and 23%, respectively, and ISH, 46.1%, 30.7%, 28.8%, and 17.3%, respectively, was higher than in control tissues, 4 and 2%, respectively (P < 0.05). SGMB count measured via quantitative PCR of SGMB DNA in terms of copy number (CN), in TU and NTU of CRC-w/bac and CRC-wo/bac, 2.96-4.72, 1.29-2.81, 2.16-2.92, and 0.67-2.07 log10 CN/g respectively, showed higher colonization in TU than in NTU and in CRC-w/bac than in CRC-wo/bac (P < 0.05). The PCR-based mRNA ratio and ISH-based percentage of positively stained cells of IL-1, 1.77 and 70.3%, COX-2, 1.63 and 44.8%, and IL-8, 1.73 and 70.3%, respectively, rather than IFN-γ, c-Myc, and Bcl-2, were higher in SGMB positive patients than in control or SGMB negative patients (P < 0.05). CONCLUSIONS: The current study indicated that colorectal cancer is remarkably associated with SGMB; moreover, molecular detection of SGMB in CRC was superior to link SGMB with CRC tumors highlighting a possible direct and active role of SGMB in CRC development through most probably inflammation-based sequel of tumor development or propagation via, but not limited to, IL-1, COX-2, and IL-8.

In vitro immunogenic and immunostimulatory effects of zwitterionized 23-valent pneumococcal polysaccharide vaccine compared with nonzwitterionized vaccine.

BACKGROUND: It was hypothesized that the observed slight immunostimulatory effect of the 23-valent pneumococcal polysaccharide (pneumo-23) vaccine might be due to the presence of low levels of zwitterionic motifs. Therefore, it was hypothesized further that introducing zwitterionic motifs experimentally into polysaccharides of pneumo-23 vaccine might render it an effective immunostimulatory agent. OBJECTIVE: This study was conducted to assess the in vitro immunostimulatory effect of zwitterionized pneumo-23 (Z-P23) vaccine compared with the nonzwitterionized commercial pneumo-23 (C-P23) vaccine. METHODS: In vitro proliferation, ELISA-based in vitro cytokine synthesis (interleukin [IL]-2, interferon [IFN]-γ, and IL-10), and immunofluorescence microscopy-based immune cell profiling (CD4(+), CD8(+), and CD21(+) cells) assays were used to evaluate the immunostimulatory effect of Z-P23 on peripheral blood mononuclear cells (PBMC) of immunosuppressed cancer (IC) patients and healthy control subjects in comparison with PBMC exposed to C-P23, concanavalin A (positive control), and phosphate-buffered saline (PBS) (negative control). RESULTS: Z-P23 induced proliferation of PBMC in the IC (81.1%) and control (75.1%) groups significantly higher than that achieved with concanavalin A in the IC group (51.0%; P = 0.01) but not in the control group (89.2%; P = NS). This was also significantly higher than that achieved with C-P23 in the IC (4.8%; P < 0.001) and control (6.2%; P < 0.001) groups. Z-P23 induced IL-2 and IFN-γ synthesis in the IC group (0.61 and 0.45 ng/mL, respectively) significantly more than that with C-P23 (0.4 and 0.45 ng/mL; P = 0.002 and P <0.001), concanavalin A (0.45 and 0.31 ng/mL; P = 0.021 and P = 0.03), and PBS (0.41 and 0.29 ng/mL; P = 0.005 and P = 0.04) but not the control group. Z-P23 induced expansion of CD4(+), CD8(+), and CD21(+) lymphocytes (39.3%, 42.7%, and 8.1%, respectively) in the IC group higher than that with C-P23 (28.3%, 30.1%, and 5.5%; P = 0.01, P = 0.003, and P = NS), concanavalin A (27.2%, 35.8%, and 4.1%; P = 0.02, P = 0.048, and P = 0.035), and PBS (25.6%, 31.9%, and 4.2%; P = 0.018, P = 0.02, and P = 0.045). CONCLUSION: The in vitro immunostimulatory potential of Z-P23 was clearly observed on PBMC of IC patients as well as, to a lesser extent, healthy control subjects, stimulating the synthesis of core cytokines of T-helper 1, and primarily inducing CD4(+) and CD8(+)T cells.

Novel Epstein-Barr virus immunoglobulin G-based approach for the specific detection of nasopharyngeal carcinoma.

PURPOSE: This study was designed to find a reliable Epstein-Barr virus (EBV) immunoglobulin (Ig) G-based diagnostic/screening test for nasopharyngeal carcinoma (NPC) able to demarcate between the NPC-related seropositivity of EBV IgG antibodies and that of other head and neck cancer (HNCA) and control groups. The NPC-associated immunosuppression affects EBV IgA much more than IgG, leading to inconsistent detection of NPC using EBV IgA antibodies. MATERIALS AND METHODS: One hundred twenty-two HNCA patients, 42 NPC, 66 laryngeal carcinoma, and 14 hypopharyngeal carcinoma and 3 groups of 100 control subjects were enrolled in this study. Enzyme-linked immunosorbent assay (ELISA) was used to find a specific cutoff value for the NPC-related seropositivity of EBV IgG antibodies. RESULTS: NPC group showed higher serum level of EBV IgG antibodies than control and other HNCA groups (P < .05). However, the traditional cutoff value, mean + 2 SDs of control subjects, failed to demarcate the seropositives of NPC patients from those of healthy population (P > .05). The new cutoff value, mean + 2 SDs of the seropositives group of control subjects who had already been grouped by the traditional cutoff value, proved successful. It succeeded to demarcate between the NPC-related EBV IgG seropositivity and that issued from the persistent, latent, or reactivated EBV infection in the population (P < .05). The sensitivity/specificity of NPC detection by the new cutoff-based ELISA kit, 76.19% and 86%, was close or higher than that of EBV IgA antibodies. CONCLUSION: EBV IgG-based ELISA could be used for the diagnosis of NPC using a new cutoff threshold that excludes the population baseline of EBV IgG seropositivity.

Investigation into the controversial association of Streptococcus gallolyticus with colorectal cancer and adenoma.

BACKGROUND: The seroprevalence of IgG antibodies of Streptococcus gallolyticus subspecies gallolyticus, CIP 105428, was evaluated to investigate the controversial association of S. gallolyticus with colorectal carcinoma and adenoma in attempt to investigate the nature of such association if any, by exploring the mRNA expression of NF-kappaB and IL-8. Moreover, the serological behavior of S. gallolyticus IgG antibodies was compared to that of an indicator bacterium of bowel, Bacteroides fragilis. METHODS: ELISA was used to measure IgG antibodies of S. gallolyticus and B. fragilis in sera of 50 colorectal cancer, 14 colorectal adenoma patients, 30 age- and sex- matched apparently healthy volunteers (HV) and 30 age- and sex- matched colonoscopically-proven tumor-free control subjects. NF-kappaB and IL-8 mRNA expression was evaluated in tumorous and non-tumorous tissue sections of carcinoma and adenoma patients in comparison with that of control subjects by using in situ hybridization assay. RESULTS: Colorectal cancer and adenoma patients were associated with higher levels of serum S. gallolyticus IgG antibodies in comparison with HV and control subjects (P < 0.05) while no similar association was found with serum IgG antibodies of B. fragilis (P > 0.05). ELISA cutoff value for the seropositivity of S. gallolyticus IgG was calculated from tumor-free control group. The expression of NF-kappaB mRNA was higher in tumorous than non-tumorous tissue sections of adenoma and carcinoma, higher in carcinoma/adenoma sections than in control subjects, higher in tumorous sections of carcinoma than in adenoma patients, and higher in S. gallolyticus IgG seropositive than in seronegative groups in both tumorous and non-tumorous sections (P < 0.05). IL-8 mRNA expression in tumorous sections of adenoma and carcinoma was higher than in non-tumorous sections, higher in carcinoma/adenoma than in control subjects, and higher in S. gallolyticus IgG seropositive than in seronegative groups in tumorous rather than non-tumorous sections (P < 0.05). CONCLUSION: S. gallolyticus most likely plays an essential role in the oncogenic progression of normal colorectal mucosa to adenoma and to CRC. This promoting/propagating role of S. gallolyticus might take place by utilizing certain inflammatory, anti-apoptotic, and angiogenic factors of transformation including NF-kappaB and IL-8.

Tumor markers of bladder cancer: the schistosomal bladder tumors versus non-schistosomal bladder tumors.

BACKGROUND: The aim of this study is to comparatively elucidate the underlying molecular pathways and clinicopathological criteria in schistosomal bladder tumor (SBT) versus non-schistosomal bladder tumor (NSBT). METHODS: This study explored the role of p53, p16, bcl-2, ki-67, c-myc, Rb and EGFR, by using Immunohistochemistry assay, in 45 SBT and 39 NSBT patients in comparison with 16 schistosomal chronic cystitis (SC), 28 non-schistosomal chronic cystitis (NSC), and 20 normal control (CTL) subjects. The studied markers in SBT and NSBT were correlated with different clinicopathological criteria namely, tumor histopathology, grading, invasiveness, stage, and presentation of the disease. RESULTS: SBT was associated with high grade invasive squamous cell carcinoma (SCC) while NSBT was associated with lower grade less invasive transitional cell carcinoma (TCC). The expression of p53, bcl-2, c-myc, and EGFR was higher in SBT than in NSBT while Rb was higher in NSBT than in SBT. However, p16 and ki-67 were not different between SBT and NSBT. The profile of molecular markers in SC was similar to NSC except for EGFR which was higher in SC than in NSC. Both SC and NSC showed higher level of p53, bcl-2, ki-67, and EGFR than in CTL group while p16, Rb, and c-myc were not different. p53 was associated with high grade SCC in both SBT and NSBT. Bcl-2 was associated with high grade invasive tumors in SBT and NSBT. P16 was associated with low grade, late stage, and recurrent SBT and high grade, invasive, late stage, and recurrent NSBT. Rb was associated with SCC in SBT, invasive tumors in NSBT, and late stage and recurrent presentation in both SBT and NSBT. C-myc was associated with high grade, invasive, and late stage SBT and SCC, high grade, invasive, and late stage NSBT. EGFR was associated with invasive SCC in SBT and invasive, high grade, and late stage TCC in NSBT. ki-67 was associated with invasive SBT and high grade late stage NSBT. CONCLUSION: SBT and NSBT showed distinct molecular profile of tumor development and progression which can be taken into consideration in fine adjusting the anti-cancer therapy for SBT and NSBT.