AIM: To investigate the anti-angiogenic potential of BZD9L1 on endothelial cells (EC) in vitro, ex vivo and in HCT116 CRC xenograft in vivo models.
METHODS: EA.hy926 EC were treated with half inhibitory concentration (IC50) (2.5 μM), IC50 (5.0 μM), and double IC50 (10.0 μM) of BZD9L1 and assessed for cell proliferation, adhesion and SIRT 1 and 2 protein expression. Next, 2.5 μM and 5.0 μM of BZD9L1 were employed in downstream in vitro assays, including cell cycle, cell death and sprouting in EC. The effect of BZD9L1 on cell adhesion molecules and SIRT 1 and 2 were assessed via real-time quantitative polymerase chain reaction (qPCR). The growth factors secreted by EC post-treatment were evaluated using the Quantibody Human Angiogenesis Array. Indirect co-culture with HCT116 CRC cells was performed to investigate the impact of growth factors modulated by BZD9L1-treated EC on CRC. The effect of BZD9L1 on sprouting impediment and vessel regression was determined using mouse choroids. HCT116 cells were also injected subcutaneously into nude mice and analyzed for the outcome of BZD9L1 on tumor necrosis, Ki67 protein expression indicative of proliferation, cluster of differentiation 31 (CD31) and CD34 EC markers, and SIRT 1 and 2 genes via hematoxylin and eosin, immunohistochemistry and qPCR, respectively.
RESULTS: BZD9L1 impeded EC proliferation, adhesion, and spheroid sprouting through the downregulation of intercellular adhesion molecule 1, vascular endothelial cadherin, integrin-alpha V, SIRT1 and SIRT2 genes. The compound also arrested the cells at G1 phase and induced apoptosis in the EC. In mouse choroids, BZD9L1 inhibited sprouting and regressed sprouting vessels compared to the negative control. Compared to the negative control, the compound also reduced the protein levels of angiogenin, basic fibroblast growth factor, platelet-derived growth factor and placental growth factor, which then inhibited HCT116 CRC spheroid invasion in co-culture. In addition, a significant reduction in CRC tumor growth was noted alongside the downregulation of human SIRT1 (hSIRT1), hSIRT2, CD31, and CD34 EC markers and murine SIRT2 gene, while the murine SIRT1 gene remained unaffected, compared to vehicle control. Histology analyses revealed that BZD9L1 at low (50 mg/kg) and high (250 mg/kg) doses reduced Ki-67 protein expression, while BZD9L1 at the high dose diminished tumor necrosis compared to vehicle control.
CONCLUSION: These results highlighted the anti-angiogenic potential of BZD9L1 to reduce CRC tumor progression. Furthermore, together with previous anticancer findings, this study provides valuable insights into the potential of BZD9L1 to co-target CRC tumor vasculatures and cancer cells via SIRT1 and/or SIRT2 down-regulation to improve the therapeutic outcome.
AIM OF THE STUDY: Recent studies have demonstrated a potent anticancer potential of P. macrocarpa, especially against HeLa cell. The objective of this study was to investigate the regulation of miRNAs on MDA-MB-231 treated with P. macrocarpa ethyl acetate fraction (PMEAF).
MATERIALS AND METHODS: The regulation of miRNAs on MDA-MB-231 cells treated with PMEAF was studied through IIlumina, Hi-Seq. 2000 platform of Next Generation Sequencing (NGS) and various in silico bioinformatics tools.
RESULTS: The PMEAF treatment against MDA-MB-231 cells identified 10 upregulated and 10 downregulated miRNAs. A set of 606 target genes of 10 upregulated miRNAs and 517 target genes of 10 downregulated miRNAs were predicted based on computational and validated databases by using miRGate DB Query. Meanwhile, results from DAVID Bioinformatics Resources 6.8 specified the functional annotation of the upregulated miRNAs involvement in cancer pathway by suppressing the oncogenes and downregulating miRNAs by expressing the tumour suppressor genes in the regulation of apoptosis pathway.
CONCLUSION: In conclusion, the results of this study proved that PMEAF is a promising anticancer agent with high cytotoxicity against MDA-MB-231 breast cancer cells and it induced apoptotic cell death mechanism through the regulation of miRNAs. PMEAF might be the best candidate for developing more potent anticancer drugs or chemo preventive supplements.
MATERIALS & METHODS: BZD9L1 was tested against metastatic CRC cell lines to evaluate cytotoxicity, cell cycle and apoptosis, senescence, apoptosis related genes and protein expressions, as well as effect against major cancer signaling pathways.
RESULTS & CONCLUSION: BZD9L1 reduced the viability, cell migration and colony forming ability of both HCT 116 and HT-29 metastatic CRC cell lines through apoptosis. BZD9L1 regulated major cancer pathways differently in CRC with different mutation profiles. BZD9L1 exhibited anticancer activities as a cytotoxic drug in CRC and as a promising therapeutic strategy in CRC treatment.
Methods: BZD9L1 and 5-FU either as single treatment or in combination were tested against CRC cells to evaluate synergism in cytotoxicity, senescence and formation of micronucleus, cell cycle and apoptosis, as well as the regulation of related molecular players. The effects of combined treatments at different doses on stress and apoptosis, migration, invasion and cell death mechanism were evaluated through two-dimensional and three-dimensional cultures. In vivo studies include investigation on the combination effects of BZD9L1 and 5-FU on colorectal tumour xenograft growth and an evaluation of tumour proliferation and apoptosis using immunohistochemistry.
Results: Combination treatments exerted synergistic reduction on cell viability on HCT 116 cells but not on HT-29 cells. Combined treatments reduced survival, induced cell cycle arrest, apoptosis, senescence and micronucleation in HCT 116 cells through modulation of multiple responsible molecular players and apoptosis pathways, with no effect in epithelial mesenchymal transition (EMT). Combination treatments regulated SIRT1 and SIRT2 protein expression levels differently and changed SIRT2 protein localization. Combined treatment reduced growth, migration, invasion and viability of HCT 116 spheroids through apoptosis, when compared with the single treatment. In addition, combined treatment was found to reduce tumour growth in vivo through reduction of tumour proliferation and necrosis compared with the vehicle control group. This highlights the potential therapeutic effects of BZD9L1 and 5-FU towards CRC.
Conclusion: This study may pave the way for use of BZD9L1 as an adjuvant to 5-FU in improving the therapeutic efficacy for the treatment of colorectal cancer.
METHODOLOGY: MCM2, 4, 5 and 7 genes expression profiles were evaluated in three cervical tissue samples each of normal cervix, human papillomavirus (HPV)-infected low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC), using Human Transcriptome Array 2.0 and validated by nCounter® PanCancer Pathway NanoString Array. Immunohistochemical expression of MCM2 protein was semi-quantitatively assessed by histoscore in tissue microarrays containing 9 cases of normal cervix, 10 LSIL, 10 HSIL and 42 cases of SCC.
RESULTS: MCM2, 4, 5 and 7 genes expressions were upregulated with increasing fold change during the progression from LSIL to HSIL and the highest in SCC. MCM2 gene had the highest fold change in SCC compared to normal cervix. Immunohistochemically, MCM2 protein was localised in the nuclei of basal cells of normal cervical epithelium and dysplastic-neoplastic cells of CIN and SCC. There was a significant difference in MCM2 protein expression between the histological groups (P = 0.039), and histoscore was the highest in HSIL compared to normal cervix (P = 0.010).
CONCLUSION: The upregulation of MCM genes expressions in cervical carcinogenesis reaffirms MCM as a proliferative marker in DNA replication pathway, whereby proliferation of dysplastic and cancer cells become increasingly dysregulated and uncontrolled. A strong expression of MCM2 protein in HSIL may aid as a concatenated screening tool in detecting pre-cancerous cervical lesions.
METHOD: Cell viability and colony formation assays were used to determine the 50% inhibitory concentration (IC50) of Et. O.s, rosmarinic acid, and gemcitabine. Different doses of gemcitabine in combination with Et. O.s or rosmarinic acid were tested against Panc-1 to select the best concentrations which possessed synergistic effects. Elucidation of molecular mechanisms responsible for mediating chemo-sensitivity in Panc-1 was performed using Quantitative Real-time PCR (QPCR), flow cytometry and immunohistochemistry.
RESULTS: Et. O.s was found to significantly sensitise Panc-1 towards gemcitabine by reducing the gene expression of multidrug-resistant protein family (MDR) (MDR-1, MRP-4, and MRP-5) and molecules related to epithelial-mesenchymal transition (ZEB-1 and Snail-1). An induction of the human equilibrate nucleoside transporter-1 (hENT-1) gene was also found in cells treated with Et. O.s-gemcitabine. The Et. O.s-gemcitabine combination induced cellular senescence, cell death and cell cycle arrest in Panc-1. In addition, the inhibition of Notch signalling was demonstrated through the downregulation of Notch 1 intracellular domain in this treatment group. In contrast, rosmarinic acid-gemcitabine combination showed no additional effects on cellular senescence, apoptosis, epithelial mesenchymal transition (EMT) markers, the MRP-4 and MRP-5 multi-drug resistance protein family, hENT-1, and the Notch pathway through Notch 1 intracellular domain.
CONCLUSION: This study provides valuable insights on the use of Et. O.s to complement gemcitabine in targeting pancreatic cancer in vitro, suggesting its potential use as a novel complementary treatment in pancreatic cancer patients.