METHODS: We investigated the anti-proliferative efficacy of polar leaf extracts (LP), non-polar leaf extracts (LN), polar stem extract (SP) and non-polar stem extracts (SN) in human breast, colorectal, lung, endometrial, nasopharyngeal, and pancreatic cancer cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assay. The most potent extracts was tested along with gemcitabine using our established drug combination analysis. The effect of the combinatory treatment in apoptosis were quantified using enzyme-linked immunosorbent assay (ELISA), Annexin V assay, antibody array and immunoblotting. Statistical significance was analysed using one-way analysis of variance (ANOVA) and post hoc Dunnett's test. A p-value of less than 0.05 (p
METHODS: We used a combination of proliferation and apoptosis assays to assess the effect of JB on AML cell lines and patient samples, with BH3 profiling being performed to identify early effects of the drug (4 h). Phosphokinase arrays were adopted to identify potential driver proteins in the cellular response to JB, the results of which were confirmed and extended using western blotting and inhibitor assays and measuring levels of reactive oxygen species.
RESULTS: AML cell growth was significantly impaired following JB exposure in a dose-dependent manner; potent colony inhibition of primary patient cells was also observed. An apoptotic mode of death was demonstrated using Annexin V and upregulation of apoptotic biomarkers (active caspase 3 and cleaved PARP). Using BH3 profiling, JB was shown to prime cells to apoptosis at an early time point (4 h) and phospho-kinase arrays demonstrated this to be associated with a strong upregulation and activation of both total and phosphorylated c-Jun (S63). The mechanism of c-Jun activation was probed and significant induction of reactive oxygen species (ROS) was demonstrated which resulted in an increase in the DNA damage response marker γH2AX. This was further verified by the loss of JB-induced C-Jun activation and maintenance of cell viability when using the ROS scavenger N-acetyl-L-cysteine (NAC).
CONCLUSIONS: This work provides the first evidence of cytotoxicity of JB against AML cells and identifies ROS-induced c-Jun activation as the major mechanism of action.
METHODS: RGCs were isolated and cultured, and monoclonal antibodies (anti-rat Thy-1, Brn3a and RBPMS) were examined by immunocytochemistry. An overexpression vector MALAT1-RNA activation (RNAa), gene knockout vector MALAT1-RNA interference (RNAi), and control vector MALAT1-negative control (NC) were constructed. A chronic high intraocular pressure (IOP) rat model of glaucoma was established by episcleral vein cauterization. The RGCs were divided into the RGC control, RGC pressure, RGC pressure + MALAT1-NC, RGC pressure + MALAT1-RNAi and RGC pressure + MALAT1-RNAa groups. Sixty Sprague-Dawley (SD) rats were randomly divided into the normal, high IOP, high IOP + MALAT1-NC, high IOP + MALAT1-RNAa and high IOP + MALAT1-RNAi groups. qRT-PCR and western blotting were used to detect the expression levels of LncRNA-MALAT1 and PI3K/Akt. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and flow cytometry were used to detect RGC apoptosis.
RESULTS: Immunocytochemistry revealed that the cultured RGCs reached 90% purity. Compared with the RGC pressure + MALAT1-NC group, the RGC pressure + MALAT1-RNAa group exhibited elevated expression levels of MALAT1, lower total protein levels of PI3K and Akt and decreased RGC apoptosis, while these expression levels were reversed in the RGC pressure + MALAT1-RNAi group. RGC numbers and PI3K/Akt expression levels in the high IOP model groups were lower than those in the normal group. In the high IOP + MALAT1-RNAa group, the mRNA and protein expression levels of PI3K/Akt were reduced but higher than those in the other three high IOP model groups. Additionally, RGC numbers in the high IOP + MALAT1-RNAa group were lower than those in the normal group but higher than those in the other three high IOP model groups.
CONCLUSION: Our study provides evidence that LncRNA-MALAT1 could inhibit RGC apoptosis in glaucoma through activation of the PI3K/Akt signaling pathway.
METHODS: This descriptive study comprised 5 patients of KCOT associated with NBCCS and 8 patients of nonsyndromic type treated in the Department of Oral Maxillofacial Surgery, Universiti Kebangsaan Malaysia Medical Centre between years 1998 and 2011. The clinical features (site, size, treatment, and recurrence), demographic characteristics, and immunohistochemistry results using antibodies of bcl-2, cyclin D1, p53, and PCNA were examined. The association of the antibody expression and the type of KCOT was analyzed using Fisher exact test.
RESULTS: Altogether there were 13 patients, 5 with syndromic KCOT (1 patient met 3 major criteria of NBCCS) and 8 with sporadic KCOT. The age range for syndromic KCT was 11 to 21 years (mean 16.00 years, SD 4.36) and 10 to 54 years (median 24.50 years, interquartile range 19.00) for the nonsyndromic KCOT. Tumor recurrence occurred in 3 patients (7.7%); 1 patient from the syndromic and 2 patients from the nonsyndromic. The most positive expression was observed in PCNA for both the syndromic and nonsyndromic samples and the least positive expression involved the p53.
CONCLUSION: PCNA, bcl-2 protein, and cyclin D1 expressions could be useful in evaluating the proliferative activity of the tumor and the aggressiveness of the clinical presentation; however, the authors would propose for larger sample size research for more definitive results.