METHODS AND RESULTS: The lactobacilli putative probiotic (SynForU-HerCare; two capsules/day of 9·5 log CFU per capsule) or placebo was administered for 8-weeks in a randomized, double-blind, placebo-controlled study. Subjects were assessed for vaginal and gut health conditions at baseline, week-4 and week-8 via questionnaires. The vulvovaginal symptom questionnaire not only covered aspects pertaining to vulvovaginal symptoms but also the quality of life impacts such as emotional, social and sexual. The administration of lactobacilli reduced symptoms of irritation (P = 0·023) and discharge (P = 0·011) starting week-4 and continued after week-8 (P
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.
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.