Association of Interferon Lambda 3 and 4 Gene SNPs and Their Expression with COVID-19 Disease Severity: A Cross-Sectional Study.

Introduction: Expression and certain SNPs of interferon lambda 3 and 4 (IFNL3 and 4) have been associated with variable outcomes in COVID-19 patients in different regions, suggesting population-specific differences in the disease outcome. This study examined the association of INFL3 and INFL4 SNPs (rs12979860 and rs368234815, respectively) and nasopharyngeal expression with COVID-19 disease severity in Pakistani patients. Methods: For this study, 117 retrospectively collected nasopharyngeal swab samples were used from individuals with mild and severe COVID-19 disease. qPCR assays were used to determine the viral loads and mRNA expression of IFNL3 and 4 through the Ct and delta Ct methods, respectively. Due to funding limitations, only one SNP each in INFL3 and INFL4 (found to be most significant through literature search) was analyzed using tetra-arm PCR and RFLP-PCR strategies, respectively. The Mann-Whitney U-test was applied to evaluate the statistical differences in the expression of IFNL3/4 genes in the mild and severe groups, while for SNPs, a Chi-square test was employed. A multivariate Cox regression test was performed to assess the relationship of different variables with COVID-19 severity. Results: Comparative analysis of SNPs between mild and severe groups showed only the difference in SNP of the IFNL4 gene to be statistically significant (p = 0.001). Similarly, nasopharyngeal expression of IFNL3 and IFNL4 genes, respectively, was found to be 3.48-fold less and 3.48-fold higher in the severe group as compared to the mild group. Multivariate analysis revealed SNP in the IFNL4 gene and age to have a significant association with COVID-19 severity. Conclusion: Despite the small sample size, IFNL4 gene SNP and patient age were associated with COVID-19 severity. Age, IFNL3/IFNL4 mRNA expression in the nasopharyngeal milieu, and the presence of SNP in the IFNL4 (rs368234815) gene in COVID-19 patients may be biomarkers for infection severity and help improve SARS-CoV-2 infection management.

XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis.

BACKGROUND: Breast cancer is the most common cancer in females and is ranked second in cancer-related deaths all over the world in women. Despite improvement in diagnosis, the survival rate of this disease has still not improved. X-linked Inhibitor of Apoptosis (XIAP) has been shown to be over-expressed in various cancers leading to poor overall survival. However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored. METHODS: We examined the expression of XIAP in more than 1000 Middle Eastern breast cancer cases by immunohistochemistry. Apoptosis was measured by flow cytometry. Protein expression was determined by western blotting. Finally, in vivo studies were performed on nude mice following xenografting and treatment with inhibitors. RESULTS: XIAP was found to be over-expressed in 29.5% of cases and directly associated with clinical parameters such as tumor size, extra nodal extension, triple negative breast cancer and poorly differentiated breast cancer subtype. In addition, XIAP over-expression was also significantly associated with PI3-kinase pathway protein; p-AKT, proliferative marker; Ki-67 and anti-apoptotic marker; PARP. XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis. Next, we investigated inhibition of XIAP using a specific inhibitor; embelin and found that embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells. Finally, breast cancer cells treated with combination of embelin and PI3-kinase inhibitor; LY294002 synergistically induced apoptosis and caused tumor growth regression in vivo. CONCLUSION: These data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells.

Co-targeting of Cyclooxygenase-2 and FoxM1 is a viable strategy in inducing anticancer effects in colorectal cancer cells.

BACKGROUND: Cross-talk between deregulated signaling pathways in cancer cells causes uncontrolled growth and proliferation. These cancers cells become more aggressive and quickly develop resistance to therapy. Therefore targeting of these deregulated pathways simultaneously can result in efficient cell death of cancer cells. In this study we investigated co-expression of Cox-2 and FoxM1 in a cohort of colorectal carcinoma (CRC) samples and also examined whether inhibition of Cox-2 and FoxM1 simultaneously can lead to inhibition of cell viability and induction of apoptosis in colorectal cancer cell lines and in vivo xenografts. METHODS: Protein expression of Cox-2 and FoxM1 was determined in a large cohort of 770 clinical CRC samples in a tissue micro-array format by immunohistochemistry. Cell death was measured using live dead assay. Apoptosis was measured by annexin V/PI dual staining. Immunoblotting was performed to examine the expression of proteins. Calcusyn software was utilized to estimate the synergistic doses using chou and Talalay method. RESULTS: Co-expression of Cox-2 and FoxM1 was detected in 33.3 % (232/697) of CRC's and associated with an aggressive phenotype characterized by younger age (p = 0.0191), high proliferative index marker; Ki-67 (p = 0.004) and MMP-9 (p = 0.0116) as well as activation of AKT (p = 0.0214). In vitro, inhibition of FoxM1 and Cox-2 with pharmacological inhibitors; Thiostrepton and NS398 resulted in efficient down-regulation of FoxM1 and Cox-2 expression along with in-activation of AKT and inhibition of colony formation, invasion and migratory capability of CRC cells. In addition, there was also inhibition of cell viability and induction of apoptosis via the mitochondrial apoptotic pathway in CRC cell lines. Finally, treatment of CRC xenograft tumors in nude mice with combination of Cox-2 and FoxM1 inhibitors inhibited tumor growth significantly via down-regulation of Cox-2 and FoxM1 expression. CONCLUSIONS: These findings demonstrate that co-expression of Cox-2 and FoxM1 might play a critical role in the pathogenesis of CRC. Therefore, targeting of these pathways simultaneously with sub toxic doses of pharmacological inhibitors can be a potential therapeutic approach for the treatment of this subset of CRC.

Dual Targeting of mTOR Activity with Torin2 Potentiates Anticancer Effects of Cisplatin in Epithelial Ovarian Cancer.

Mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) are two key components of PI3K/Akt/mTOR signaling pathway. Dysregulation of these pathways have been found in many cancers, including epithelial ovarian cancer (EOC), however, the role of mTOR has not been fully elucidated in Middle Eastern EOC. Therefore, we investigated the activation of mTOR complexes (mTORC1 and mTORC2) in a cohort of 156 EOC from Saudi Arabia by immunohistochemistry in a tissue microarray format. mTORC1 and mTORC2 were found to be activated in 55 of 146 (37.7%) and 63 of 140 (45%) of EOC samples, respectively. mTORC1 was significantly associated with mTORC2 (p < 0.0001) activation and both mTOR complexes were significantly associated with p-AKT (p = 0.0205 and 0.0298) and p-P70S6 (p < 0.0001 and 0.0035), respectively. Interestingly, mTOR activation incurred a poor progression-free survival (PFS) (p = 0.0188) in EOC. Next, the in vitro effect of inactivation of mTOR complexes was evaluated using a second-generation mTOR inhibitor, Torin2, on a panel of EOC cell lines. Torin2 treatment decreased cell viability and induced apoptosis in a dose-dependent manner via inactivation of mTORC1 and mTORC2 and their downstream targets in EOC cell lines. Furthermore, treatment of EOC cells with a subtoxic dose of Torin2 potentiated a cisplatin-induced apoptotic response in EOC cell lines. Finally, we studied the in vivo effect of a combination of Torin2 and cisplatin and found that this combination synergistically inhibited tumor growth in nude mice. These studies highlight the importance of targeting the mTOR survival pathway and suggest that cotreatment with cisplatin and Torin2 may be beneficial for the management of EOC.

Role of X-Linked Inhibitor of Apoptosis as a Prognostic Marker and Therapeutic Target in Papillary Thyroid Carcinoma.

CONTEXT: Papillary thyroid cancer (PTC) is the second most common cancer in females in Saudi Arabia. However, the pathogenesis of PTC is still not fully elucidated. OBJECTIVE: To identify potential genes that play important role in progression of PTC, we studied the role of X-linked inhibitor of apoptosis protein (XIAP) as a potential prognostic marker and therapeutic target in a large cohort of PTC samples and cell lines. DESIGN: A DNA microarray chip was used to screen for gene copy number. XIAP expression was assessed by immunohistochemistry in a tissue microarray format on a cohort of 1022 clinical samples. In vitro and in vivo studies were performed using Embelin and/or LY294002 on PTC cell lines. RESULTS: XIAP was found to be amplified in 14 of 29 and overexpressed in 48.8% of PTC cases. XIAP overexpression was significantly associated with old age, extrathyroidal extension, tumor size, nodal involvement, tall-cell variant, advanced stage disease, and significantly poor disease-free survival (P = .0341). XIAP was also significantly associated with phosphorylated AKT (P < .0001), Bcl-Xl (P < .0001), and Ki67 (P = .0006) proteins. Embelin treatment caused growth inhibition and apoptosis in PTC cell lines and induced tumor regression in PTC xenograft in nude mice. Finally, the combination of suboptimal doses of Embelin and LY294002 induced a synergistic apoptotic response in PTC cells. CONCLUSION: XIAP dysregulation in PTC confers an aggressive phenotype with poor outcome. In vitro and in vivo studies using an XIAP inhibitor suggest that this subgroup of PTC with overexpression of XIAP can be therapeutically targeted, either alone or in combination, to induce efficient apoptosis in these cancers.

High prevalence of mTOR complex activity can be targeted using Torin2 in papillary thyroid carcinoma.

The mammalian target of rapamycin (mTOR) signaling cascade is a key regulatory pathway controlling initiation of messenger RNA in mammalian cells. Although dysregulation of mTOR signaling has been reported earlier in cancers, there is paucity of data about mTOR expression in papillary thyroid carcinoma (PTC). Therefore, in this study, we investigated the presence of mTORC2 and mTORC1 complexes in a large cohort of >500 PTC samples. Our clinical data showed the presence of active mTORC1 and mTORC2 in 81 and 39% of PTC samples, respectively. Interestingly, coexpression of mTORC1 and mTORC2 activity was seen in a 32.5% (164/504) of the PTC studied and this association was statistically significant (P = 0.0244). mTOR signaling complex was also found to be associated with activated AKT and 4E-BP1. In vitro, using Torin2, a second-generation mTOR inhibitor or gene silencing of mTOR expression prevented mTORC1 and mTORC2 activity leading to inactivation of P70S6, 4E-BP1, AKT and Bad. Inhibition of mTOR activity led to downregulation of cyclin D1, a gene regulated by messenger RNA translation via phosphorylation of 4E-BP1. Torin2 treatment also inhibited cell viability and induced caspase-dependent apoptosis via activation of mitochondrial apoptotic pathway in PTC cells. Finally, Torin2 treatment induces anticancer effect on PTC xenograft tumor growth in nude mice via inhibition of mTORC1 and mTORC2 and its associated pathways. Our results suggest that coexpression of mTORC1 and mTORC2 is seen frequently in the clinical PTC samples and dual targeting of mTORC1 and mTORC2 activity may be an attractive therapeutic target for treatment of PTC.

Bortezomib inhibits proteasomal degradation of IκBα and induces mitochondrial dependent apoptosis in activated B-cell diffuse large B-cell lymphoma.

Activated B-cell type lymphoma (ABC), a subgroup of diffuse large B-cell lymphoma (DLBCL), has a worse survival after upfront chemotherapy and is characterized by constitutive activation of the anti-apoptotic nuclear factor-κB (NFκB) pathway. The implication of NFκB inhibition in ABC has not yet been fully explored as a potential therapeutic target. Therefore, a panel of ABC cell lines was used to examine the effect of bortezomib, a proteasome inhibitor which blocks degradation of IκBα and consequently inhibits NFκB activity. Our data showed that bortezomib caused a dose-dependent growth inhibition and induction of apoptosis in all cell lines studied. We next determined the status of the NFκB pathway following bortezomib treatment and found that there was accumulation of IκBα without affecting its phosphorylation status at an early time point. Electrophoretic mobility shift assay showed that bortezomib treatment inhibited constitutive nuclear NFκB in ABC cell lines. Furthermore, treatment of ABC cell lines with bortezomib for 48 h also down-regulated the expression of NFκB-regulated gene products, such as IκBα, Bcl-2, Bcl-Xl, XIAP and survivin, leading to apoptosis via the mitochondrial apoptotic pathway. Altogether, these results suggest that NFκB may be a potential target for therapeutic intervention in DLBCL using proteasomal inhibitors such as bortezomib.

Role of dysregulated expression of leptin and leptin receptors in colorectal carcinogenesis.

Leptin is a multifunctional adipose-derived cytokine that plays a critical role in bodyweight homeostasis and energy balance. Plasma level of leptin is an indicator of the amount of energy stored in adipose tissues. Recently, leptin and leptin receptor dysregulation have been reported in a variety of malignant cells including colorectal cancers (CRCs). There are growing evidence that leptin may be the link between obesity and CRC carcinogenesis. Leptin influence the growth and proliferation of cancer cells via activation of various growth and survival signaling pathways including JAK/STAT, PI3-kinase/AKT, and/or MAP kinases. In this review, current understanding of leptin and its receptor's roles in the pathogenesis of colonogenic cancer has been described.

Phosphorylated IκBα predicts poor prognosis in activated B-cell lymphoma and its inhibition with thymoquinone induces apoptosis via ROS release.

Activated B-cell lymphoma (ABC), one of the three subtypes of Diffuse Large B-cell Lymphoma (DLBCL) has the worst survival rate after upfront chemotherapy and is characterized by constitutively activated NFκB. We therefore studied the role of NFκB In a cohort of clinical DLBCL samples and ABC cell lines. In our clinical tissue microarray cohort of DLBCL samples, p-IκBα was detected in 38.3% of ABC DLBCL and was an independent prognostic marker for poor survival. In vitro, we found that Thymoquinone (TQ), a natural compound isolated from Nigella sativa caused release of ROS in ABC cells. TQ-mediated release of ROS in turn inhibited NFκB activity by dephosphorylating IκBα and decreased translocation of p65 subunit of NFκB in the nuclear compartment in ABC cell lines. This led to inhibition of cell viability and induction of mitochondrial dependent apoptosis in ABC-DLBCL cell lines. Additionally, TQ treatment also caused up-regulation of death receptor 5 (DR5), however, up-regulation of DR5 did not play a role in TQ-induced apoptosis. Finally, combination of sub-optimal doses of TQ and TRAIL induced efficient apoptosis in ABC-DLBCL cell lines. These data show that p-IκBα can be used as a prognostic marker and target for therapy in this aggressive sub-type of DLBCL and TQ may play an important role in the management of DLBCL in the future.

Cross-talk between NFkB and the PI3-kinase/AKT pathway can be targeted in primary effusion lymphoma (PEL) cell lines for efficient apoptosis.

BACKGROUND: A number of constitutively activated signaling pathways play critical roles in the survival and growth of primary effusion lymphoma cells (PELs) including NFkB and PI3/AKT kinase cascades. NFkBis constitutively activated in a number of malignancies, including multiple myeloma, Burkitt's lymphoma and diffuse large cell B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored. METHODOLOGY/PRINCIPAL FINDINGS: We used pharmacological inhibition and gene silencing to define the role of NFkB in growth and survival of PEL cells. Inhibition of NFkB activity by Bay11-7085 resulted in decreased expression of p65 in the nuclear compartment as detected by EMSA assays. In addition, Bay11-7085 treatment caused de-phosphorylation of AKT and its downstream targets suggesting a cross-talk between NFkB and the PI3-kinase/AKT pathway. Importantly, treatment of PEL cells with Bay11-7085 led to inhibition of cell viability and induced apoptosis in a dose dependent manner. Similar apoptotic effects were found when p65 was knocked down using specific small interference RNA. Finally, co-treatment of PEL cells with suboptimal doses of Bay11-7085 and LY294002 led to synergistic apoptotic responses in PEL cells. CONCLUSION/SIGNIFICANCE: These data support a strong biological-link between NFkB and the PI3-kinase/AKT pathway in the modulation of anti-apoptotic effects in PEL cells. Synergistic targeting of these pathways using NFKB- and PI3-kinase/AKT-inhibitors may have a therapeutic potential for the treatment of PEL and possibly other malignancies with constitutive activation of these pathways.

Overexpression of FoxM1 offers a promising therapeutic target in diffuse large B-cell lymphoma.

BACKGROUND: FoxM1 has been shown to play a critical role in the pathogenesis of various epithelial malignancies. However, its role in lymphoid malignancies has not been fully clarified. We, therefore, investigated the role of FoxM1 expression in a large cohort of diffuse large B-cell lymphoma samples and panel of cell lines. DESIGN AND METHODS: FoxM1 expression was investigated in a large series of diffuse large B-cell lymphoma tissues in a tissue microarray format by immunohistochemistry. Apoptosis was measured by flow cytometry and protein expression was detected by immunoblotting using diffuse large B-cell lymphoma cell lines following treatment with either pharmacological inhibitor of FoxM1 or small interference RNA knockdown strategy. Invasion/migration and soft agar colony assays were also performed following treatment with FoxM1 inhibitor. RESULTS: FoxM1 expression was detected in 84.6% of diffuse large B-cell lymphoma tumors and found to be significantly associated with proliferative tumor marker Ki67 (P<0.0001), matrix metalloproteinases-2 (P=0.0008), matrix metalloproteinases-9 (P=0.0002), S-phase kinase associated protein-2 (P<0.0001) and inversely associated with p27 expression (P=0.0215). Expression of small interference RNA targeted against FoxM1 or treatment of diffuse large B-cell lymphoma cells with thiostrepton caused its downregulation accompanied by decreased expression of matrix metalloproteinases-2 and matrix metalloproteinases-9. Inhibition of FoxM1 in diffuse large B-cell lymphoma cells also decreased invasive and migratory capability, and induced caspase dependent apoptosis via activation of the mitochondrial apoptotic pathway. Finally, combined thiostrepton and bortezomib at sub-toxic doses led to efficient apoptosis in diffuse large B-cell lymphoma cells. CONCLUSIONS: Altogether, these results suggest that FoxM1 is over-expressed in the majority of diffuse large B-cell lymphoma samples. These data also indicate that targeting FoxM1 signaling can serve as a potential therapeutic modality in the management of diffuse large B-cell lymphoma.

c-Met inhibitor synergizes with tumor necrosis factor-related apoptosis-induced ligand to induce papillary thyroid carcinoma cell death.

The Met receptor tyrosine kinase is overexpressed and/or activated in variety of human malignancies. Previously we have shown that c-Met is overexpressed in Middle Eastern papillary thyroid carcinoma (PTC) and significantly associated with an aggressive phenotype, but its role has not been fully elucidated in PTC. The aim of this study was to determine the functional link between the c-Met/AKT signaling pathway and death receptor 5 (DR5) in a large cohort of PTC in a tissue microarray format followed by functional studies using PTC cell lines and nude mice. Our data showed that high expressions of p-Met and DR5 were significantly associated with an aggressive phenotype of PTC and correlated with BRAF mutation. Treatment of PTC cell lines with PHA665752, an inhibitor of c-Met tyrosine kinase, inhibited cell proliferation and induced apoptosis via the mitochondrial pathway in PTC cell lines. PHA665752 treatment or expression of c-Met small interfering (si)RNA resulted in dephosphorylation of c-Met, AKT and its downstream effector molecules. Furthermore, PHA665752 treatment upregulated DR5 expression via generation of reactive oxygen species in PTC cell lines, and synergistically potentiated death receptor-induced apoptosis with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Finally, cotreatment with PHA665752 and TRAIL caused more pronounced effects on PTC xenograft tumor growth in nude mice. Our data suggest that the c-Met/AKT pathway may be a potential target for therapeutic intervention for treatment of PTC refractory to conventionally therapeutic modalities.

FoxM1 and its association with matrix metalloproteinases (MMP) signaling pathway in papillary thyroid carcinoma.

CONTEXT: Forkhead boxM1 (FoxM1) transcription factor has been shown to promote pathogenesis of several malignancies. FoxM1 has also been shown to be associated with matrix metalloproteinases (MMP) in various cancers. However, little is known about its function in papillary thyroid carcinoma (PTC). OBJECTIVE: In this study, we investigated the role of FoxM1 in pathogenesis in a large series of PTC in a tissue microarray format followed by in vitro and in vivo studies using PTC cell lines and nude mice. DESIGN: Expression of FoxM1 and its associated proteins were investigated in Middle Eastern PTC samples by immunohistochemistry. Apoptosis was measured by flow cytometry and immunoblotting. Invasion and migration studies were performed using 8-µm Transwell plates. RESULTS: FoxM1 was overexpressed in 28.4% of PTC and significantly associated with activated matrix metalloproteinase-9 (MMP-9) (P = 0.0004), X-linked inhibitor of apoptosis protein (XIAP) (P = 0.0024), and B-cell lymphoma-extra large (Bcl-XL) (P = 0.0014) expression. Treatment of PTC cell lines with thiostrepton, an inhibitor of FoxM1, resulted in inhibition of cell viability via induction of apoptosis. In addition, thiostrepton treatment of PTC cells or expression of FoxM1-specific small interfering RNA down-regulated expression of FoxM1 accompanied with decreased MMP-2 and MMP-9 expression. Furthermore, inhibition of FoxM1 attenuated migration and invasion of PTC cells. Interestingly, overexpression of FoxM1 rescued the effects of thiostrepton in PTC cell lines. Finally, treatment of PTC cell line xenografts with thiostrepton resulted in growth inhibition of tumors in nude mice via down-regulation of FoxM1 and MMP-9 and MMP-2. CONCLUSION: Altogether, this is the first study showing that FoxM1 and its associated signaling pathway play a critical role in the pathogenesis of PTC and may be a potential target for therapeutic intervention for treatment of these cancers.

Role of leptin and its receptors in the pathogenesis of thyroid cancer.

Leptin is a multifunctional adipose-derived cytokines that play a critical role in bodyweight homeostasis and energy balance. Recently, leptin and leptin receptor dysreulation have been reported in variety of malignant cells including thyroid. Leptin modulates growth and proliferation of cancer cells via activation of various growth and survival signaling pathways including JAK/STAT, PI3-kinase/AKT and/or Map kinases. In this review, current understanding of leptin's role in the pathogenesis of thyroid cancer has been described.

Coexpression of activated c-Met and death receptor 5 predicts better survival in colorectal carcinoma.

Dysregulated overexpression of hepatocyte growth factor and its receptor, c-Met, has been reported in various cancers, but its role in colorectal carcinoma (CRC) has not been elucidated. Therefore, we investigated the role of phosphorylated Met (p-Met) in Middle Eastern CRC patient samples and cell lines. The p-Met was overexpressed in 80.8% of CRCs and strongly associated with the expression of p-AKT, DR5, and Ki-67 by immunohistochemistry. Coexpression of p-Met and DR5 was seen in 53.1% of CRC cases and was associated with a less aggressive phenotype, characterized by a histological subtype of adenocarcinomas, well-differentiated tumors, and was an independent prognostic marker for better overall survival. PHA665752, a selective p-Met inhibitor, induced apoptosis in CRC cells via inactivation of c-Met and AKT. PHA665752 treatment also caused increased expression of DR5 via generation of reactive oxygen species, and combination treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and PHA665752 induced significant apoptosis. In vivo, cotreatment of a CRC xenograft with PHA665752 and TRAIL significantly reduced tumor volume and weight. These data demonstrate a significant correlation between p-Met and DR5 in patients with CRC. Furthermore, inhibition of p-Met signaling by PHA665752 in combination with TRAIL significantly inhibited cell growth and induced apoptosis in CRC cell lines, suggesting that this may have significant clinical implications as a therapeutic target in the treatment of CRC.

Resveratrol suppresses constitutive activation of AKT via generation of ROS and induces apoptosis in diffuse large B cell lymphoma cell lines.

BACKGROUND: We have recently shown that deregulation PI3-kinase/AKT survival pathway plays an important role in pathogenesis of diffuse large B cell lymphoma (DLBCL). In an attempt to identify newer therapeutic agents, we investigated the role of Resveratrol (trans-3,4', 5-trihydroxystilbene), a naturally occurring polyphenolic compound on a panel of diffuse large B-cell lymphoma (DLBCL) cells in causing inhibition of cell viability and inducing apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the action of Resveratrol on DLBCL cells and found that Resveratrol inhibited cell viability and induced apoptosis by inhibition of constitutively activated AKT and its downstream targets via generation of reactive oxygen species (ROS). Simultaneously, Resveratrol treatment of DLBCL cell lines also caused ROS dependent upregulation of DR5; and interestingly, co-treatment of DLBCL with sub-toxic doses of TRAIL and Resveratrol synergistically induced apoptosis via utilizing DR5, on the other hand, gene silencing of DR5 abolished this effect. CONCLUSION/SIGNIFICANCE: Altogether, these data suggest that Resveratrol acts as a suppressor of AKT/PKB pathway leading to apoptosis via generation of ROS and at the same time primes DLBCL cells via up-regulation of DR5 to TRAIL-mediated apoptosis. These data raise the possibility that Resveratrol may have a future therapeutic role in DLBCL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.

The biological and clinical impact of inhibition of NF-κB-initiated apoptosis in diffuse large B cell lymphoma (DLBCL).

NF-κB is frequently over-expressed in a variety of non-Hodgkin's lymphomas (NHLs) and has been implicated in lymphomagenesis; however, its role in diffuse large B cell lymphoma (DLBCL) as a prognostic biomarker has not been fully elucidated. Therefore, we investigated the role of NF-κB and its association with clinicopathological features in a tissue microarray cohort of 230 DLBCL patient samples. We then elucidated the role of NF-κB inhibition on cell viability and apoptosis in vitro, using DLBCL cell lines. Using immunohistochemistry, NF-κB was detected in 25.6% (52/203) DLBCL tumours, was associated with activated B cell (ABC) phenotype (p = 0.0054), Epstein-Barr virus (EBV; p = 0.0080) and over-expression of the anti-apoptotic marker XIAP (p = 0.0013). DLBCL cases with nuclear expression of NF-κB showed a significantly poorer overall survival as compared to those without NF-κB expression (p = 0.0236). In a multivariate analysis using a Cox proportional hazard model for IPI and NF-κB expression, the relative risk was 2.97 for high NF-κB expression (95% CI 1.27-6.94; p = 0.0113) and 7.55 for the high-IPI group (95% CI 3.34-18.35; p < 0.0001). In vitro, Bay 11-7085 inhibited constitutively active NF-κB expression in a dose-dependent manner and inhibition of NF-κB also down-regulated expression of the downstream target gene products Bcl-2, Bcl-XL (BCL2L1), XIAP and Survivin, leading to apoptosis via activation of the mitochondrial apoptotic pathway. NF-κB over-expression was found to be an independent prognostic marker for poor survival in DLBCL. Altogether, these results suggest that NF-κB may be a useful prognostic biomarker and a potential target for therapeutic intervention in DLBCL.

Bortezomib stabilizes mitotic cyclins and prevents cell cycle progression via inhibition of UBE2C in colorectal carcinoma.

Substantial evidence implicates the ubiquitin-conjugating enzyme E2C (UBE2C) gene, in several human cancers, including colorectal carcinoma (CRC). We therefore investigated the prognostic value of UBE2C alterations in CRC and UBE2C signaling in CRC cell lines. UBE2C protein expression and UBE2C gene copy number were evaluated on clinical samples by immunohistochemistry and fluorescence in situ hybridization in a TMA format. The effect of the proteasome inhibitor bortezomib and small-interfering RNA knockdown was assessed by apoptotic assays and immunoblotting. UBE2C dysregulation was associated with proliferative marker Ki-67, accumulation of cyclin A and B1, and a poor overall survival. UBE2C expression was an independent prognostic marker in early-stage (I and II) CRC. UBE2C depletion resulted in suppression of cellular growth and accumulation of cyclin A and B1. In vitro, bortezomib treatment of CRC cells caused inhibition of cell viability via down-regulation of UBE2C. UBE2C knockdown by bortezomib or transfection with specific small-interfering RNA against UBE2C also caused cells to be arrested at the G2/M level, leading to accumulation of cyclin A and cyclin B1. In vivo, a significant reduction in tumor volume and weight was noted in mice treated with a combination of subtoxic doses of oxaliplatin and bortezomib compared with treatment with oxaliplatin or bortezomib alone. Altogether, our results suggest that UBE2C and the ubiquitin-proteasome pathway may be potential targets for therapeutic intervention in CRC.

Thymoquinone suppresses growth and induces apoptosis via generation of reactive oxygen species in primary effusion lymphoma.

We provide evidence that thymoquinone (TQ), a natural compound isolated from Nigella sativa, induces growth inhibition and apoptosis in several primary effusion lymphoma (PEL) cell lines. Our data demonstrate that TQ treatment results in down-regulation of constitutive activation of AKT via generation of reactive oxygen species (ROS) and it causes conformational changes in Bax protein, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. This leads to activation of caspase-9, caspase-3, and polyadenosine 5'-diphosphate ribose polymerase cleavage, leading to caspase-dependent apoptosis. Pretreatment of PEL cells with N-acetylcysteine, a scavenger of ROS, prevented TQ-mediated effects. In addition, subtoxic doses of TQ sensitized PEL cells to TRAIL via up-regulation of DR5. Altogether, these findings demonstrate that TQ is a potent inducer of apoptosis in PEL cells via release of ROS. They also raise the possibility that incorporation of TQ in treatment regimens for primary effusion lymphomas may provide a novel approach to sensitizing malignant cells and provide a molecular basis for such future translational efforts.

HGF/c-Met pathway has a prominent role in mediating antiapoptotic signals through AKT in epithelial ovarian carcinoma.

The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are overexpressed and/or activated in a variety of human malignancies. However, its role in epithelial ovarian carcinoma (EOC) has not been clearly elucidated. Therefore, we investigated the role of HGF/c-Met signaling pathway in a large series (156) of Saudi EOC patient samples, a panel of cell lines, and xenografts in a NUDE mouse model. Using immunohistochemistry, c-Met overexpression was found in 27.2% Middle Eastern EOC samples and was associated with an advanced tumor stage (P=0.0187). c-Met overexpression was also associated with antiapoptotic markers X-chromosome-linked inhibitors of apoptosis (XIAP) (P=0.0008) and Bcl-XL (P=0.0493) expression. Treatment of EOC cell lines with PHA665752 causes a dose-dependent inhibition of cell viability and induction of apoptosis. Furthermore, PHA665752 treatment causes dephosphorylation of AKT and downregulation of antiapoptotic proteins XIAP and Bcl-XL. In addition, PHA665752-induced apoptosis occurs through activation of Bax-mediated release of cytochrome c and activation of caspases. Finally, co-treatment of EOC with PHA665752 and cisplatin causes augmented effect on apoptosis of EOC cells and resulted in synergistic inhibition of EOC xenograft tumor growth in NUDE mice. These results indicate that c-Met/HGF pathway may be a potential target for therapeutic intervention for treatment of EOC.