Probiotics have been reported to ameliorate symptoms of type 2 diabetes mellitus (T2DM) in animal models and human studies. We previously demonstrated that oral administration of Lactobacillus reuteri ADR-3 reduced insulin resistance in high-fructose-fed (HFD) rats. In the present study, we first identified another L. reuteri strain, ADR-1, which displayed anti-diabetes activity that reduced the levels of serum HbA1c and cholesterol and that increased antioxidant proteins in HFD rats. We further performed a randomized, double-blinded, placebo-controlled trial with a total of 68 T2DM patients to examine the beneficial effects of oral consumption of L. reuteri strains ADR-1 and ADR-3 and to investigate the associated changes in intestinal flora using a quantitative PCR method to analyze 16 S rRNA in fecal specimens. Significant reductions in HbA1c and serum cholesterol were observed in participants in the live ADR-1 consumption group (n = 22) after 3 months of intake when compared with those in the placebo group (n = 22). Although there was no significant difference in the HbA1c serum level among participants who consumed heat-killed ADR-3 (n = 24), the systolic blood pressure and mean blood pressure were significantly decreased after 6 months of intake. There was no obvious change in serum inflammatory cytokines or antioxidant proteins in participants after intaking ADR-1 or ADR-3, except for a reduction in IL-1β in the ADR-3 consumption group after 6 months of intake. With the analysis of fecal microflora, we found that L. reuteri or Bifidobacterium spp. were significantly increased in the ADR-1 and ADR-3 consumption groups, respectively, after 6 months of intake. Interestingly, a significant reduction in HbA1c was observed in the ADR-1 and ADR-3 consumption participants who displayed at least an 8-fold increase in fecal L. reuteri. We also observed that there was a significantly positive correlation between Bifidobacterium spp. and Lactobacillus spp. in participants with increased levels of fecal L. reuteri. In the ADR-1 intake group, the fecal Lactobacillus spp. level displayed a positive correlation with Bifidobacterium spp. but was negatively correlated with Bacteroidetes. The total level of fecal L. reuteri in participants in the ADR-3 consumption group was positively correlated with Firmicutes. In conclusion, L. reuteri strains ADR-1 and ADR-3 have beneficial effects on T2DM patients, and the consumption of different strains of L. reuteri may influence changes in intestinal flora, which may lead to different outcomes after probiotic intake.

Tumorsphere as an effective in vitro platform for screening anti-cancer stem cell drugs

To improve prognosis in recurrent glioblastoma we developed a treatment protocol based on a combination of drugs not traditionally thought of as cytotoxic chemotherapy agents but that have a robust history of being well-tolerated and are already marketed and used for other non-cancer indications. Focus was on adding drugs which met these criteria: a) were pharmacologically well characterized, b) had low likelihood of adding to patient side effect burden, c) had evidence for interfering with a recognized, well-characterized growth promoting element of glioblastoma, and d) were coordinated, as an ensemble had reasonable likelihood of concerted activity against key biological features of glioblastoma growth. We found nine drugs meeting these criteria and propose adding them to continuous low dose temozolomide, a currently accepted treatment for relapsed glioblastoma, in patients with recurrent disease after primary treatment with the Stupp Protocol. The nine adjuvant drug regimen, Coordinated Undermining of Survival Paths, CUSP9, then are aprepitant, artesunate, auranofin, captopril, copper gluconate, disulfiram, ketoconazole, nelfinavir, sertraline, to be added to continuous low dose temozolomide. We discuss each drug in turn and the specific rationale for use- how each drug is expected to retard glioblastoma growth and undermine glioblastoma’s compensatory mechanisms engaged during temozolomide treatment. The risks of pharmacological interactions and why we believe this drug mix will increase both quality of life and overall survival are reviewed.

We examined the expression in breast cancer stem cells (BCSCs) of Globo H, a potential tumor-associated antigen for immunotherapy of epithelial cancers including breast cancer. Flow cytometry revealed Globo H expression in 25/41 breast cancer specimens (61.0%). Non-BCSCs from 25/25 and BCSCs from 8/40 (20%) expressed Globo H. We showed the expression of stage-specific embryonic antigen 3 (SSEA3), the pentasaccharide precursor of Globo H, in 31/40 (77.5%) tumors. Non-BCSCs from 29/31 and BCSCs from 25/40 (62.5%) expressed SSEA3. Like Globo H, SSEA3 expression in normal tissues was predominately at the secretory borders of epithelium, where access to the immune system is restricted. Immunization of mice with Globo H-KLH and α-GalCer induced antibodies reactive with Globo H and SSEA3, suggesting that a Globo H-based vaccine will target tumor cells expressing Globo H or SSEA3. We next sought to reduce Globo H expression by siRNA targeting fucosyltransferase ( FUT ) 1 and 2, which mediate alpha-1,2 linkage of fucose to SSEA3 to generate Globo H. We showed both genes to be involved in the biosynthesis of Globo H. Moreover, FUT2 expression in BCSCs was significantly lower than in non-BCSCs harvested from a primary human breast cancer in NOD/SCID mouse, whereas FUT1 was slightly lower in BCSCs. Thus, the lower expression of Globo H in BCSCs may be attributed to less FUT2/FUT1, and to reduced SSEA3 in BCSCs compared with non-BCSCs. Our findings provide insight into further development of a Globo H-based vaccine and FUT1/FUT2 -targeted therapy for breast cancer.

Introduction: Heat shock proteins (HSPs) are normally induced under environmental stress to serve as chaperones for maintenance of correct protein folding but they are often overexpressed in many cancers, including breast cancer. The expression of Hsp27, an ATP-independent small HSP, is associated with cell migration and drug resistance of breast cancer cells. Breast cancer stem cells (BCSCs) have been identified as a subpopulation of breast cancer cells with markers of CD24-CD44+ or high intracellular aldehyde dehydrogenase activity (ALDH+) and proved to be associated with radiation resistance and metastasis. However, the involvement of Hsp27 in the maintenance of BCSC is largely unknown. Methods: Mitogen-activated protein kinase antibody array and Western blot were used to discover the expression of Hsp27 and its phosphorylation in ALDH + BCSCs. To study the involvement of Hsp27 in BCSC biology, siRNA mediated gene silencing and quercetin treatment were used to inhibit Hsp27 expression and the characters of BCSCs, which include ALDH+ population, mammosphere formation and cell migration, were analyzed simultaneously. The tumorigenicity of breast cancer cells after knockdown of Hsp27 was analyzed by xenograftment assay in NOD/SCID mice. The epithelial-mesenchymal transition (EMT) of breast cancer cells was analyzed by wound-healing assay and Western blot of snail, vimentin and E-cadherin expression. The activation of nuclear factor kappa B (NF-κB) was analyzed by luciferase-based reporter assay and nuclear translocation. Results: Hsp27 and its phosphorylation were increased in ALDH+ BCSCs in comparison with ALDH- non-BCSCs. Knockdown of Hsp27 in breast cancer cells decreased characters of BCSCs, such as ALDH+ population, mammosphere formation and cell migration. In addition, the in vivo CSC frequency could be diminished in Hsp27 knockdown breast cancer cells. The inhibitory effects could also be observed in cells treated with quercetin, a plant flavonoid inhibitor of Hsp27, and it could be reversed by overexpression of Hsp27. Knockdown of Hsp27 also suppressed EMT signatures, such as decreasing the expression of snail and vimentin and increasing the expression of E-cadherin. Furthermore, knockdown of Hsp27 decreased the nuclear translocation as well as the activity of NF-κB in ALDH + BCSCs, which resulted from increasing expression of IκBα. Restored activation of NF-κB by knockdown of IκBα could reverse the inhibitory effect of Hsp27 siRNA in suppression of ALDH+ cells. Conclusions: Our data suggest that Hsp27 regulates the EMT process and NF-κB activity to contribute the maintenance of BCSCs. Targeting Hsp27 may be considered as a novel strategy in breast cancer therapy.

Oral submucous fibrosis (OSF) is considered as a pre‐cancerous condition of the oral mucosa and is highly associated with habitual areca quid chewing. Arecoline is the major alkaloid in areca quid and is thought to be involved in the pathogenesis of OSF. Our previous studies have demonstrated that arecoline could induce epithelial–mesenchymal transition (EMT)‐related factors in primary human buccal mucosal fibroblasts (BMFs). Therefore, we investigated the expression of zinc finger E‐box binding homeobox 1 (ZEB1), which is a well‐known transcriptional factor in EMT, in OSF tissues and its role in arecoline‐induced myofibroblast transdifferentiation from BMFs. The expression of ZEB1, as well as the myofibroblast marker α‐smooth muscle actin (α‐SMA), was significantly increased in OSF tissues, respectively. With immunofluorescence analysis, arecoline induced the formation of α‐SMA‐positive stress fibres in BMFs expressing nuclear ZEB1. Arecoline also induced collagen contraction of BMFs in vitro. By chromatin immunoprecipitation, the binding of ZEB1 to the α‐SMA promoter in BMFs was increased by arecoline. The promoter activity of α‐SMA in BMFs was also induced by arecoline, while knockdown of ZEB1 abolished arecoline‐induced α‐SMA promoter activity and collagen contraction of BMFs. Long‐term exposure of BMFs to arecoline induced the expression of fibrogenic genes and ZEB1. Silencing of ZEB1 in fibrotic BMFs from an OSF patient also suppressed the expression of α‐SMA and myofibroblast activity. Inhibition of insulin‐like growth factor receptor‐1 could suppress arecoline‐induced ZEB1 activation in BMFs. Our data suggest that ZEB1 may participate in the pathogenesis of areca quid–associated OSF by activating the α‐SMA promoter and inducing myofibroblast transdifferentiation from BMFs.