Omar Al-Obeed

New targeted-colon delivery system: in vitro and in vivo evaluation using X-ray imaging

The aim of this study was to formulate a new orally-administered colon delivery system of 5-flurouracil (5-FU) for the treatment of colon cancer. The system was designed to target 5-FU directly to the colon with high potential of much more effective and less toxic colon cancer treatment. The system was prepared by compression coating technique using granulated chitosan. The method was optimized by studying the effect of granulation and thickness of the coat with respect to the in vitro performance in a medium mimicking mouth-to-colon environment. The in vivo selectivity of the system was assessed by X-ray imaging technique using beagle dogs. Results showed that granulation of chitosan were effective in protecting against the known acid solubility of the polymer. Formula (F7) with coat weight of  50 mg/tablet exhibited the best protection profile with <10% of the drug released after 6 h. The resistance of the system to the simulated gastro-intestinal media was reduced as the chitosan coat weight decreases. The performance of the system in a rat caecal contents containing-medium showed that the susceptibility of this system for the enzymatic degradation by colonic enzymes. The X-ray imaging gave rise to the in vivo selectivity of this system for colon targeting by showing the resistivity of the system to the stomach and small intestine environment and the selective disintegration of the system inside the large bowel.

Design, synthesis and in vitro evaluation of anticancer and antibacterial potential of surface modified Tb(OH)3@SiO2 core–shell nanoparticles

In the current study, we modified the surface of Tb(OH)3 nanoparticles with a silica layer to enhance their solubility and biocompatibility. Transmission electron microscopy confirmed the improvements in these properties. Tb(OH)3@SiO2 core–shell nanoparticles (TS-CSNPs) exhibited a strong green emission peak upon irradiation with ultraviolet light, which originates from the electric-dipole transition 5D4 → 7F5 (543 nm) of the Tb3+ ion. In vitro anticancer and antimicrobial activities of the synthesized TS-CSNPs has been evaluated through their potential cytotoxicity and antibacterial activity. TS-CSNPs were shown to have more cytotoxicity against HT29 human colorectal cancer cells with a value of IC50 420.33 in an MTT assay. The alteration of the morphological features of HT29 cells was analysed using various concentrations of TS-CSNPs by inverted microscopy. Western blot analysis results of the apoptotic pathway showed that TS-CSNPs inhibited the growth of HT29 cancer cells through the induction of apoptosis, as evidenced by the down regulation of the expression of anti-apoptotic Bcl-2 and Bcl-xL gene products. Furthermore, the results of the in vitro hemolysis assay with human erythrocytes demonstrated the excellent blood biocompatibility of TS-CSNPs. Our silica coated TS-CSNPs exhibited a non-significant effect on the viability of both Gram negative and Gram positive bacterial strains up to 18 hours of exposure. These results highlight that modified TS-CSNPs can be functionalized to enhance the efficacy of cancer therapeutics due to the significant potential against cancerous cells and antibacterial activity.

Novel derivative of aminobenzenesulfonamide (3c) induces apoptosis in colorectal cancer cells through ROS generation and inhibits cell migration

BackgroundColorectal cancer (CRC) is the 3rd most common type of cancer worldwide. New anti-cancer agents are needed for treating late stage colorectal cancer as most of the deaths occur due to cancer metastasis. A recently developed compound, 3c has shown to have potent antitumor effect; however the mechanism underlying the antitumor effect remains unknown.Methods3c-induced inhibition of proliferation was measured in the absence and presence NAC using MTT in HT-29 and SW620 cells and xCELLigence RTCA DP instrument. 3c-induced apoptotic studies were performed using flow cytometry. 3c-induced redox alterations were measured by ROS production using fluorescence plate reader and flow cytometry and mitochondrial membrane potential by flow cytometry; NADPH and GSH levels were determined by colorimetric assays. Bcl2 family protein expression and cytochrome c release and PARP activation was done by western blotting. Caspase activation was measured by ELISA. Cell migration assay was done using the real time xCELLigence RTCA DP system in SW620 cells and wound healing assay in HT-29.ResultsMany anticancer therapeutics exert their effects by inducing reactive oxygen species (ROS). In this study, we demonstrate that 3c-induced inhibition of cell proliferation is reversed by the antioxidant, N-acetylcysteine, suggesting that 3c acts via increased production of ROS in HT-29 cells. This was confirmed by the direct measurement of ROS in 3c-treated colorectal cancer cells. Additionally, treatment with 3c resulted in decreased NADPH and glutathione levels in HT-29 cells. Further, investigation of the apoptotic pathway showed increased release of cytochrome c resulting in the activation of caspase-9, which in turn activated caspase-3 and −6. 3c also (i) increased p53 and Bax expression, (ii) decreased Bcl2 and BclxL expression and (iii) induced PARP cleavage in human colorectal cancer cells. Confirming our observations, NAC significantly inhibited induction of apoptosis, ROS production, cytochrome c release and PARP cleavage. The results further demonstrate that 3c inhibits cell migration by modulating EMT markers and inhibiting TGFβ-induced phosphorylation of Smad2 and Samd3.ConclusionsOur findings thus demonstrate that 3c disrupts redox balance in colorectal cancer cells and support the notion that this agent may be effective for the treatment of colorectal cancer.

Public awareness of colorectal cancer in Saudi Arabia: A survey of 1070 participants in Riyadh.

Background/Aims: The aim of this study was to investigate colorectal cancer (CRC) awareness in healthy individuals in Saudi Arabia in order to identify segments of the population that would most benefit from targeted education programs. Setting and Design: Survey/questionnaire. Patients and Methods: Random, healthy individuals from Riyadh, Saudi Arabia, were approached to participate in a 10-question multiple choice survey about CRC. Data were analyzed by demographic criteria, including age, gender, marital status, and level of education, to determine if members of these groups displayed differential knowledge. Statistical Analysis: Differences in responses by demographic data were analyzed using Pearsons Chi-square test. A P < 0.05 was considered statistically significant. Results: In total, 1070 participants completed the survey. Most respondents believe that screening for colon cancer should begin at symptom onset (42.9%). Less than 20% of all respondents believe that polyps are a risk factor for CRC, which varied significantly according to level of education; however, even the most educated answered correctly less than 50% of the time. Similarly, only 34.8% of all respondents knew that a family history of CRC imparted a personal risk for CRC. Conclusions: Although older individuals and those with higher education tended to answer questions correctly more often, there were some misconceptions regarding universally accepted screening protocols, symptoms, and general understanding of CRC in Saudi Arabia. A national education/screening program in Saudi Arabia is recommended to improve CRC knowledge.

Cathepsin B expression in colorectal cancer in a Middle East population: Potential value as a tumor biomarker for late disease stages

Cathepsin B (CTSB), is a cysteine protease belonging to the cathepsin (Clan CA) family. The diagnostic and prognostic significance of increased CTSB in the serum of cancer patients have been evaluated for some tumor types. CTSB serum and protein levels have also been reported previously in colorectal cancer (CRC) with contradictory results. The aim of the present study was to investigate CTSB expression in CRC patients and the association of CTSB expression with various tumor stages in a Middle East population. Serum CTSB levels were evaluated in 70 patients and 20 healthy control subjects using enzyme-linked immunosorbant assay (ELISA) technique. CTSB expression was determined in 100 pairs of CRC tumor and adjacent normal colonic tissue using quantitative PCR for mRNA levels. Detection of CTSB protein expression in tissues was carried out using both immunohistochemistry and western blotting techniques. ELISA analysis showed that in sera obtained from CRC patients, the CTSB concentration was significantly higher in late stage patients with lymph node metastases when compared to early stage patients with values of 2.9 and 0.33 ng/ml, respectively (P=0.001). The majority of tumors studied had detectable CTSB protein expression with significant increased positive staining in tumors cells when compared with matched normal colon subjects (P=0.006). The mRNA expression in early stage CRC compared to late stage CRC was 0.04±0.01 and 0.07±0.02, respectively. Increased mRNA expression was more frequently observed in the advanced cancer stages with lymph node metastases when compared with the control (P=0.002). Mann-Whitney test and paired t-test were used to compare serum CTSB and mRNA levels in early and late tumor stage. A subset of four paired tissue extracts were analyzed by western blotting. The result confirmed a consistent increase in the CTSB protein expression level in tumor tissues compared with that noted in the adjacent normal mucosal cells. These findings indicate that CTSB may be an important prognostic biomarker for late stage CRC and cases with lymph node metastases in the Middle Eastern population. Monitoring serum CTSB in CRC patients may predict and/or diagnose cases with lymph node metastases.

Novel quinazoline-based sulfonamide derivative (3D) induces apoptosis in colorectal cancer by inhibiting JAK2–STAT3 pathway

Introduction Colorectal cancer (CRC) is a major worldwide health problem owing to its high prevalence and mortality rate. Developments in screening, prevention, biomarker, personalized therapies and chemotherapy have improved detection and treatment. However, despite these advances, many patients with advanced metastatic tumors still succumb to the disease. New anticancer agents are needed for treating advanced stage CRC as most of the deaths occur due to cancer metastasis. A recently developed novel sulfonamide derivative 4-((2-(4-(dimethylamino) phenyl)quinazolin-4-yl)amino)benzenesulfonamide (3D) has shown potent antitumor effect; however, the mechanism underlying the antitumor effect remains unknown. Materials and methods 3D-mediated inhibition on cell viability was evaluated by MTT and real-time cell proliferation was measured by xCelligence RTDP instrument. Western blotting was used to measure pro-apoptotic, anti-apoptotic proteins and JAK2-STAT3 phosphorylation. Flow cytometry was used to measure ROS production and apoptosis. Results Our study revealed that 3D treatment significantly reduced the viability of human CRC cells HT-29 and SW620. Furthermore, 3D treatment induced the generation of reactive oxygen species (ROS) in human CRC cells. Confirming our observation, N-acetylcysteine significantly inhibited apoptosis. This is further evidenced by the induction of p53 and Bax; release of cytochrome c; activation of caspase-9, caspase-7 and caspase-3; and cleavage of PARP in 3D-treated cells. This compound was found to have a significant effect on the inhibition of antiapoptotic proteins Bcl2 and BclxL. The results further demonstrate that 3D inhibits JAK2–STAT3 pathway by decreasing the constitutive and IL-6-induced phosphorylation of STAT3. 3D also decreases STAT3 target genes such as cyclin D1 and survivin. Furthermore, a combination study of 3D with doxorubicin (Dox) also showed more potent effects than single treatment of Dox in the inhibition of cell viability. Conclusion Taken together, these findings indicate that 3D induces ROS-mediated apoptosis and inhibits JAK2–STAT3 signaling in CRC.

TLTF in cerebrospinal fluid for detection and staging of T. b. gambiense infection.

Background Trypanosome-derived lymphocyte triggering factor (TLTF) is a molecule released by African trypanosomes that interacts with the host immune system, resulting in increased levels of IFN-γ production. Methodology/Principal findings TLTF and anti-TLTF antibodies were assessed in sera and cerebrospinal fluid (CSF) from patients infected with Trypanosoma brucei gambiense (T. b. gambiense) in an attempt to identify alternative markers for diagnosis and stage determination of human African trypanosomiasis or sleeping sickness. Seventy-four serum and sixty-one CSF samples from patients with parasitologically confirmed infection and known disease stage along with 13 sera and CSF from uninfected controls were tested. In serum the levels of anti-TLTF antibodies were unrelated to the disease stage. In contrast, levels of anti-TLTF antibodies in CSF were higher in intermediate/late stages than in early stage disease patients. Specificity of the detected antibodies was assessed by inhibition of TLTF bioactivity as represented by its ability to induce IFN-γ production. Additionally, TLTF was detected in CSF from late stage patients by Western blotting with the anti-TLTF specific monoclonal antibody MO3. Conclusions/Significance These findings suggest a new possibility for disease diagnosis with focus on involvement of the CNS through detection of TLTF and anti-TLTF antibodies in the CSF.

Abstract 2160: Novel sulfonamide derivative inhibits JAK2-STAT3 signaling and induces ROS-mediated apoptosis in colorectal cancer cells

Colorectal cancer is a major worldwide health problem owing to its high prevalence and mortality rate. Developments in screening, prevention, biomarker, personalized therapies and chemotherapy have improved detection and treatment. However, despite these advances many patients with advanced metastatic tumors will still succumb to the disease. New anti-cancer agents are needed for treating advance stage colorectal cancer as most of the deaths occur due to cancer metastasis. A recently developed novel sulphonamide derivative, 4-((2-(4-(Dimethylamino)phenyl)quinazolin-4-yl)amino)benzenesulfonamide (3d) has shown to have potent antitumor effect; however the mechanism underlying the antitumor effect remains unknown. Our study revealed that 3d treatment significantly reduced the viability of human colorectal cancer cells HT-29 and SW620. This is further evidenced by the induction of p53 and Bax, release of cytochrome c, activation of caspase-9, caspase-7 and caspase-3 and cleavage of PARP in 3d treated cells. This compound was found to have significant effect on the inhibition of anti-apoptotic proteins, Bcl2, BclxL and XIAP. The results further demonstrate that 3d inhibited JAK2-STAT3 pathway by decreasing the constitutive and IL-6-induced phosphorylation of JAK2 and STAT3. 3d further decreased JAK2- STAT3 target genes like Cyclin D1 and Survivin. Furthermore 3d treatment induced the generation of reactive oxygen species (ROS) in human colorectal cancer cells. Confirming our observation, NAC significantly inhibited ROS production, induction of apoptosis, cytochrome c release and PARP cleavage. Additionally, treatment with 3d resulted in decreased glutathione (GSH) levels. The results further demonstrate that 3d inhibited cell migration by modulating EMT markers and inhibiting TGFβ-induced phosphorylation of Smad2 and Samd3. Collectively these findings indicate that 3d inhibited JAK2-STAT3 signaling; induced apoptosis via generation of reactive oxygen species and inhibition of cell migration by altering EMT markers and TGFβ-Smad pathway. Note: This abstract was not presented at the meeting. Citation Format: Rehan Ahmad, Mansoor-Ali Vaali-Mohammed, Omar Al-Obeed, Maha Abdulla. Novel sulfonamide derivative inhibits JAK2-STAT3 signaling and induces ROS-mediated apoptosis in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2160. doi:10.1158/1538-7445.AM2017-2160