The murine double minute 2 (MDM2) protein is a major negative regulator of the tumour suppressor protein p53. Under normal conditions, MDM2 constantly binds to p53 transactivation domain and/or ubiquinates p53 via its role as E3 ubiquitin ligase to promote p53 degradation as well as nuclear export to maintain p53 levels in cells. Meanwhile, amplification of MDM2 and appearance of MDM2 spliced variants occur in many tumours and normal tissues making it a prognostic indicator for human cancers. The mutation or deletion of p53 protein in half of human cancers inactivates its tumour suppressor activity. However, cancers with wild type p53 have its function effectively inhibited through direct interaction with MDM2 oncoprotein. Here, we described the construction of a MDM2 spliced variant (rMDM215kDa) consisting of SWIB/MDM2 domain and its central region for antibody generation. Biopanning with a human naïve scFv library generated four scFv clones specific to rMDM215kDa. Additionally, the selected scFv clones were able to bind to the recombinant full length MDM2 (rMDM2-FL). Computational prediction showed that the selected scFv clones potentially bind to exon 7-8 of MDM2 while leaving the MDM2/SWIB domain free for p53 interaction. The developed antibodies exhibit good specificity can be further investigated for downstream biomedical and research applications.
Single Stock Futures (SSFs) was introduced in Bursa Malaysia on 28th April 2006. There have been many studies on derivative instruments in Malaysia; however, none is on SSFs. Various statistical methods have been used to analyse the SSFs and its spot returns, namely Descriptive Statistics, Unit Root test, VAR, Johansen and Juselius Co-integration test, Granger Causality test, Variance Decomposition test, VECM, and GARCH model. This study analyses the SSFs and spot returns of eight companies listed in Bursa Malaysia. It found that Berjaya Sports Toto Bhd and Genting Bhd have no long-run and short-run causality (Genting Bhd has bi-directional causality) while AirAsia Bhd and AMMB Holdings Bhd’s spot returns’ volatility decreased after the introduction of SSFs; it increased in the other seven companies. In addition, only AMMB Holdings Bhd futures return did not affect its spot return. Bursa Malaysia Bhd and RHB Capital Bhd spot returns lead their futures returns
Protective action by annatto-derived delta-tocotrienol (δ-TCT) and soy-derived alpha-tocopherol (α-TOC) through the regulation of PI3K/Akt-Cyclin D1 pathway against the nicotine-induced DNA damages is the focus of the present study. Nicotine, which has been widely reported to have numerous adverse effects on the reproductive system, was used as reproductive toxicant. 48 female balb/c mice (6-8 weeks) (23-25 g) were randomly divided into 8 groups (G1-G8; n = 6) and treated with either nicotine or/and annatto δ-TCT/soy α-TOC for 7 consecutive days. On Day 8, the females were superovulated and mated before euthanized for embryo collection (46 hours post-coitum). Fifty 2-cell embryos from each group were used in gene expression analysis using Affymetrix QuantiGene Plex2.0 assay. Findings indicated that nicotine (G2) significantly decreased (p < 0.05) the number of produced 2-cell embryos compared to control (G1). Intervention with mixed annatto δ-TCT (G3) and pure annatto δ-TCT (G4) significantly increased the number of produced 2-cell embryos by 127 % and 79 % respectively compared to G2, but these were lower than G1. Concurrent treatment with soy α-TOC (G5) decreased embryo production by 7 %. Supplementations with δ-TCT and α-TOC alone (G6-G8) significantly increased (p < 0.05) the number of produced 2-cell embryos by 50 %, 36 % and 41 % respectively, compared to control (G1). These results were found to be associated with the alterations in the PI3K/Akt-Cyclin D1 gene expressions, indicating the inhibitory effects of annatto δ-TCT and soy α-TOC against the nicotinic embryonic damages. To our knowledge, this is the first attempt on studying the benefits of annatto δ-TCT on murine preimplantation 2-cell embryos.
Amniotic fluid stem cells (AFSCs) are commonly isolated from mid-term amniotic fluid (AF) of animals and human collected via an invasive technique, amniocentesis. Alternatively, AFSCs could be collected at full-term. However, it is unclear whether AFSCs are present in the AF at full term. Here, we aimed to isolate and characterize stem cells isolated from AF of full term pregnant rats. Three stem cell lines have been established following immuno-selection against the stem cell marker, c-kit. Two of the new lines expressed multiple markers of pluripotency until more than passage 90. Further, they spontaneously differentiated into derivatives of the three primary germ layers through the formation of good quality embryoid bodies (EBs), and can be directly differentiated into neural lineage. Their strong stemness and potent neurogenic properties highlight the presence of highly potent stem cells in AF of full-term pregnancies, which could serve as a potential source of stem cells for regenerative medicine.
Annexin A1 (ANXA1) is an endogenous protein involved in the control of proliferation, cell cycle, phagocytosis, and apoptosis in several types of cancer. To investigate the effects of ANXA1 knockdown in leukemia cells, transfection with specific ANXA1 siRNA was performed. Cell cycle and apoptosis were analyzed using flow cytometry and a mechanism involving caspases and Bcl-2 was quantified using Western blotting. Phagocytosis activity was evaluated using hematoxylin & eosin staining. The ANXA1 expression was significantly downregulated after the knockdown and apoptosis was induced in tested cells. The expression of caspase-9 and -3 increased in U937 and Jurkat cells respectively. Bcl-2 expression was downregulated in K562 and Jurkat cells while upregulated in U937. The number of leukemic cells arrested at the G2/M phase and the phagocytosis index were significantly increased in transfected cells. This suggests that ANXA1 knockdown might be a potential approach in the therapeutic strategy for leukemia.
Colorectal cancer (CRC) is often caused by mutations in the KRAS oncogene, making KRAS neoantigens a promising vaccine candidate for immunotherapy. Secreting KRAS antigens using live Generally Recognized as Safe (GRAS) vaccine delivery hosts such as Lactococcus lactis is deemed to be an effective strategy in inducing specific desired responses. Recently, through the engineering of a novel signal peptide SPK1 from Pediococcus pentosaceus, an optimized secretion system was developed in the L. lactis NZ9000 host. In this study, the potential of the L. lactis NZ9000 as a vaccine delivery host for the production of two KRAS oncopeptides (mutant 68V-DT and wild-type KRAS) through the use of the signal peptide SPK1 and its mutated derivative (SPKM19) was investigated. The expression and secretion efficiency analyses of KRAS peptides from L. lactis were performed in vitro and in vivo in BALB/c mice. Contradictory to our previous study using the reporter staphylococcal nuclease (NUC), the yield of secreted KRAS antigens mediated by the target mutant signal peptide SPKM19 was significantly lower (by ~1.3-folds) compared to the wild-type SPK1. Consistently, a superior elevation of IgA response against KRAS aided by SPK1 rather than mutant SPKM19 was observed. Despite the lower specific IgA response for SPKM19, a positive IgA immune response from mice intestinal washes was successfully triggered following immunization. Size and secondary conformation of the mature proteins are suggested to be the contributing factors for these discrepancies. This study proves the potential of L. lactis NZ9000 as a host for oral vaccine delivery due to its ability to evoke the desired mucosal immune response in the gastrointestinal tract of mice.
The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression.
SIGNIFICANCE: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis.
Follicular lymphoma is frequently associated with t(14;18)(q32;q21) translocation. This study was undertaken to determine the pattern of Bcl-2, CD10 and Bcl-6 expression in relation to t(14;18) translocation in follicular lymphoma from a cohort of a multi-ethnic Asian population.
Imatinib mesylate is an anti‑neoplastic targeted chemotherapeutic agent, which can inhibit tyrosine kinase receptors, including BCR‑ABL, platelet‑derived growth factor receptors (PDGFRs) and c‑Kit. Cellular processes, including differentiation, proliferation and survival are regulated by these receptors. The present study aimed to evaluate the antiproliferative effects of imatinib mesylate, and its effects on apoptotic induction and cell cycle arrest in breast cancer cell lines. In addition, the study aimed to determine whether the effects of this drug were associated with the mRNA and protein expression levels of PDGFR‑β, c‑Kit, and their corresponding ligands PDGF‑BB and stem cell factor (SCF), which may potentially modulate cell survival and proliferation. To assess the antiproliferative effects of imatinib mesylate, an MTS assay was conducted following treatment of cells with 2‑10 µM imatinib mesylate for 96, 120 and 144 h; accordingly the half maximal inhibitory concentration of imatinib mesylate was calculated for each cell line. In addition, the proapoptotic effects and cytostatic activity of imatinib mesylate were investigated. To evaluate the expression of imatinib‑targeted genes, PDGFR‑β, c‑Kit, PDGF‑BB and SCF, under imatinib mesylate treatment, mRNA expression was detected using semi‑quantitative polymerase chain reaction and protein expression was detected by western blot analysis in ZR‑75‑1 and MDA‑MB‑231 breast carcinoma cell lines. Treatment with imatinib mesylate suppressed cell proliferation, which was accompanied by apoptotic induction and cell cycle arrest in the investigated cell lines. In addition, PDGFR‑β, PDGF‑BB, c‑Kit and SCF were expressed in both breast carcinoma cell lines; PDGFR‑β and c‑Kit, as imatinib targets, were downregulated in response to imatinib mesylate treatment. The present results revealed that at least two potential targets of imatinib mesylate were expressed in the two breast carcinoma cell lines studied. In conclusion, the antiproliferative, cytostatic and proapoptotic effects of imatinib mesylate may be the result of a reduction in the expression of c‑Kit and PDGFR tyrosine kinase receptors, thus resulting in suppression of the corresponding ligand PDGF‑BB. Therefore, imatinib mesylate may be considered a promising target therapy for the future treatment of breast cancer.
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play a critical role in transmitting signals from cell-surface molecules to intracellular protein effectors. Key PI3Ks include PI3Kα, PI3Kβ, and PI3Kδ, which are regulated by receptors. The signaling pathway comprising the PI3Ks, along with a Ser/Thr kinase (AKT), a proto-oncogene product (mouse double minute (MDM)2), and a tumor suppressor protein (p53), plays an essential role in experimental proliferative vitreoretinopathy (PVR), which is a fibrotic blinding eye disorder. However, which PI3K isoforms are involved in PVR is unknown. A major characteristic of PVR is the formation of epi (or sub)-retinal membranes that consist of extracellular matrix and cells, including retinal pigment epithelium (RPE) cells, glial cells, and macrophages. RPE cells are considered key players in PVR pathogenesis. Using immunoblotting and immunofluorescence analyses, we herein provide the evidence that PI3Kδ is highly expressed in human RPEs when it is primarily expressed in leukocytes. We also found that PI3Kδ inactivation through two approaches, CRISPR/Cas9-mediated depletion and a PI3Kδ-specific inhibitor (idelalisib), not only blocks vitreous-induced activation of AKT and MDM2 but also abrogates a vitreous-stimulated decrease in p53. Furthermore, we demonstrate that PI3Kδ inactivation prevents vitreous-induced proliferation, migration, and contraction of human RPEs. These results suggest that PI3Kδ may represent a potential therapeutic target for RPE-related eye diseases, including PVR.
The enhancement of cell proliferation and promotion of cell survival via the inhibition of apoptosis is thought to be the key to the initiation and progression of cancers. The phosphatidylinositol-3 kinase (PI3K)/Akt is an important survival signal pathway that has been shown to be crucial in the regulation of balance between pro-apoptotic and survival (anti-apoptotic) signal. In this study, the expression of phosphorylated Akt at Thr308 and Ser473, BCL-2-antagonist of cell death (BAD) at Ser136 and glycogen synthase kinase-3beta (GSK-3beta) at Ser9 in 47 paraffin-embedded human colorectal carcinoma (CRC) tissues were determined by immunohistochemical staining in order to dissect the alterations in the signal transduction pathways in CRC. Our results showed that there was a significant increase in the expression of these biomolecules in CRC tissues compared to the apparently normal adjacent tissues. The frequency of increased expression in tumor colonic mucosa were as follows: p-Akt1/2/3 (Thr308) = 16/47 (34%); p-Akt1 (Ser473) = 21/47 (44.7%); phospho-BAD (p-BAD) Ser136 = 27/47 (57.4%) and phospho-GSK-3beta (p-GSK-3beta) = 21/47 (44.7%). Analysis of the total p-Akt1 (Ser473), p-Akt1/2/3 (Thr308), p-GSK-3beta (Ser9) and p-BAD (Ser136) score found that there was a statistically significant relationship with each other. A statistically significant positive linear relationship was found between total p-Akt (Ser473) score and total p-GSK-3beta (Ser9) score as well as with total p-BAD (Ser136) score. On the other hand, total p-Akt1/2/3 (Thr308) scores had a statistically significant positive linear relationship with p-GSK-3beta (Ser9) only. The Akt targets, p-GSK-3beta (Ser9) and p-BAD (Ser136) were positively correlated to each other. There was no significant correlation between clinico-pathological data with total p-Akt1 (Ser473), p-Akt1/2/3 (Thr308), p-GSK-3beta (Ser9) and p-BAD (Ser136) score except for age. The total scores of p-GSK-3beta were found to be higher in patients in the age group of greater than 60. This is the first report of p-Akt1/2/3 (Thr308) and p-BAD (Ser136) expression in primary colorectal tumor tissue. Our data further supports the role of PI3K/Akt signaling pathways in the pathogenesis of CRC and contributes to the identification of target molecules in the signal transduction pathway for cancer therapy.
Jatropha meal produced from the kernel of Jatropha curcas Linn. grown in Malaysia contains phorbol esters (PEs). The potential benefits of PEs present in the meal as anticancer agent are still not well understood. Hence, this study was conducted to evaluate the cytotoxic effects and mode of actions of PEs isolated from Jatropha meal against breast (MCF-7) and cervical (HeLa) cancer cell lines. Isolated PEs inhibited cells proliferation in a dose-dependent manner of both MCF-7 and HeLa cell lines with the IC₅₀ of 128.6 ± 2.51 and 133.0 ± 1.96 µg PMA equivalents/mL respectively, while the values for the phorbol 12-myristate 13-acetate (PMA) as positive control were 114.7 ± 1.73 and 119.6 ± 3.73 µg/mL, respectively. Microscopic examination showed significant morphological changes that resemble apoptosis in both cell lines when treated with PEs and PMA at IC₅₀ concentration after 24 h. Flow cytometry analysis and DNA fragmentation results confirmed the apoptosis induction of PEs and PMA in both cell lines. The PEs isolated from Jatropha meal activated the PKC-δ and down-regulated the proto-oncogenes (c-Myc, c-Fos and c-Jun). These changes probably led to the activation of Caspase-3 protein and apoptosis cell death occurred in MCF-7 and HeLa cell lines upon 24 h treatment with PEs and PMA. Phorbol esters of Jatropha meal were found to be promising as an alternative to replace the chemotherapeutic drugs for cancer therapy.
Chemostat cultures of NS0 cell lines were carried out at dilution rates ranging from 0.8 d(-1) to 0.2 d(-1). Compared with the control, the viable cell density of the Bcl-2 cell line was approximately 10% higher at 0.8 d(-1) and increased to 55% when the dilution rate was reduced to 0.2 d(-1). As the dilution rate was reduced, the viability of the two cultures diverged reaching a difference of 43% at 0.2 d(-1). The specific growth rate of the control cells was the same as the dilution rate down to a value of 0.6 d(-1). By contrast, the specific growth rate of Bcl-2 cells was parallel to the dilution rate down to a value as low as 0.3 d(-1). For both NS0 cell lines, the G1 cell population decreased, while the S and G2/M cell populations increased as the dilution rate was reduced. The antibody titer of the control cells increased from 7 to 21 microg.ml(-1) as the dilution rate was reduced from 0.8 to 0.2 d(-1). With an initial increase from 2 to 15 microg.ml(-1) as the dilution rate was reduced from 0.8 to 0.4 d(-1), the antibody titer of the Bcl-2 cells remained constant as the dilution rate was further reduced to 0.2 d(-1). A good correlation between specific antibody production rate and the percentage of G2/M cells was observed.
Xanthorrhizol is a sesquiterpenoid compound extracted from Curcuma xanthorrhiza, which is known locally as Temulawak. Traditionally, C. xanthorrhiza was found to have antibacterial, anticancer and anti-inflammatory activity. The rhizome has also been used to treat inflammation in postpartum uterine bleeding. An antiproliferative assay using methylene blue staining revealed that xanthorrhizol inhibited the proliferation of the cervical cancer cell line HeLa with an EC50 value of 6.16 microg/ml. Xanthorrhizol significantly increased apoptosis in HeLa cells, as evaluated by the Tdt-mediated dUTP nick end-labelling (TUNEL) assay and nuclear morphology by Hoechst 33258 staining. Western blot analysis, which was further confirmed by the immunostaining results, implied an up-regulation of tumor suppressor protein p53 and the pro-apoptotic protein Bax, following the treatment with xanthorrhizol. Xanthorrhizol, however, did not affect the expression of the anti-apoptotic protein, Bcl-2 and the viral oncoprotein, E6. Hence, xanthorrhizol is a promising antiproliferative and anticancer agent which induces p53 and Bax-dependent apoptosis in HeLa cervical cancer cells.
Bcl-2 family proteins are crucial regulators of apoptosis. Both pro- and antiapoptotic members exist, and overexpression of the latter facilitates evasion of apoptosis in many cancer types. Bcl-2 homology domain 3 (BH3) mimetics are small molecule inhibitors of antiapoptotic Bcl-2 family members, and these inhibitors are promising anticancer agents. In this study, we report that gamma-tocotrienol (γT3), an isomer of vitamin E, can inhibit Bcl-2 to induce apoptosis. We demonstrate that γT3 induces cell death in human neuroblastoma SH-SY5Y cells by depolarising the mitochondrial membrane potential, enabling release of cytochrome c to the cytosol and increasing the activities of caspases-9 and -3. Treatment of cells with inhibitors of Bax or caspase-9 attenuated the cell death induced by γT3. Simulated docking analysis suggested that γT3 binds at the hydrophobic groove of Bcl-2, while a binding assay showed that γT3 competed with a fluorescent probe to bind at the hydrophobic groove. Our data suggest that γT3 mimics the action of BH3-only protein by binding to the hydrophobic groove of Bcl-2 and inducing apoptosis via the intrinsic pathway in a Bax- and caspase-9-dependent manner.
The molecular events that drive the progression of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) are still to be elucidated. Here, we report for the first time the pathogenic significance of an NPC-associated gene, wingless-type MMTV integration site family, member 5A (WNT5A) and the contribution of EBV to its expression. WNT5A is a representative Wnt protein that activates non-canonical Wnt signalling. With regard to its role in carcinogenesis, there is conflicting evidence as to whether WNT5A has a tumour-promoting or tumour-suppressive role. We show that WNT5A is upregulated in primary NPC tissue samples. We also demonstrate that WNT5A expression was dramatically increased in NPC cell lines expressing the EBV-encoded LMP2A gene, suggesting that this EBV-encoded latent gene is responsible for upregulating WNT5A in NPC. In addition, in vitro WNT5A overexpression promotes the proliferation, migration and invasion of NPC cells. Our results not only reveal pro-tumorigenic effects of WNT5A in NPC but also suggest that WNT5A could be an important therapeutic target in patients with EBV-associated disease.
The hallmark of follicular lymphoma is the t(14;18)(q32;q21) chromosomal translocations that lead to deregulation of BCL2 expression in tumour cells. However, not all cases of follicular lymphoma express BCL2, nor is the t(14;18) translocation always present. Follicular lymphomas lacking the BCL2 rearrangement are less well studied with regards to their immunohistochemical and molecular features. This study aims to investigate the BCL2 protein expression pattern in t(14;18) negative follicular lymphomas.
The efficacy of cisplatin for treating nasopharyngeal carcinoma (NPC) is limited by the dose-related toxicities and the development of resistance to cisplatin. Recent studies have shown that B cell lymphoma-2 (BCL-2) is overexpressed and confers chemoresistance in NPC. Thus, targeted therapy against BCL-2 may enhance the antitumour effects of chemotherapy by sensitizing the tumor cells to undergo apoptosis. This study evaluated the combined effects of BCL-2 inhibition and cisplatin in NPC cells. Our results demonstrate that inhibition of BCL-2 by small-hairpin RNA (shRNA) or the BCL-2 inhibitor YC137, synergizes cisplatin sensitivity in NPC cells that overexpress BCL-2. We also show that YC137 enhance cisplatin-induced apoptosis in HK1 and CNE1 cells through suppression of BCL-2 protein expression, induction of mitochondrial depolarization and activation of caspase 9 and caspase 3/7. These findings suggest that the combination of BCL-2 inhibition and cisplatin represents a promising strategy for treating NPC.
Lung cancer remains a major health problem with a low 5-year survival rate of patients. Recent studies have shown that dysregulation of microRNAs (miRNAs) are prevalent in lung cancer and these aberrations play a significant role in the progression of tumour progression. In the present study, bioinformatics analyses was employed to predict potential miR-608 targets, which are associated with signaling pathways involved in cancer. Luciferase reporter assay identified AKT2 as a novel target of miR-608, and suppression of its protein levels was validated through western blot analysis. Zebrafish embryos were microinjected with cells transfected with miR-608 to elucidate the role of miR-608 in vivo, and immunostained with antibodies to detect activated caspase-3. We present the first evidence that miR-608 behaves as a tumour suppressor in A549 and SK-LU-1 cells through the regulation of AKT2, suggesting that selective targeting of AKT2 via miR-608 may be developed as a potential therapeutic strategy for miRNA-based non-small cell lung cancer (NSCLC) therapy.