Ground glass hepatocytes (GGHs) are the historic hallmarks for the hepatocytes in the late and non-replicative stages of hepatitis B virus (HBV) infection. We have identified type I and type II GGHs that contain two mutant types of large HBV surface antigens (HBsAg) with deletions over the pre-S1 and pre-S2 regions, respectively. These pre-S mutant HBVsAg accumulate in endoplasmic reticulum (ER), resulting in strong ER stress. Type II GGHs often appear in hepatic nodules in the late phases of HBV infection and proliferate in clusters, suggesting that these mutant pre-S1/S2 HBsAg may be involved in HBV-related hepatocarcinogenesis, associated with ER stress. In this study, we investigated the potential genomic instability imposed by pre-S mutant HBsAg. Based on the analysis of comet assays, we found that the pre-S1 and pre-S2 mutant HBsAg caused oxidative stress and DNA damage. The DNA repair gene ogg1 was greatly induced by over-expression of pre-S mutant HBsAg. Induction of the DNA repair gene ogg1 was also detected in the pre-S2 HBsAg transgenic mice, as well as the type II GGHs from patients with hepatocellular carcinoma, strongly suggesting that the pre-S mutant HBsAg contributes to the oxidative DNA damage to hepatocytes. In addition, the mutation rates in the X-linked hprt gene were enhanced in mouse hepatoma ML1-4a cells, which constitutively expressed the pre-S1/S2 HBsAg. These results indicate that pre-S1/S2 mutant HBsAg, which make up GGHs, induce oxidative DNA damage and mutations in hepatocytes in the late stages of HBV infection.

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.

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.

Cancer stem cells (CSCs) are a sub-population of cells within cancer tissues with tumor initiation, drug resistance and metastasis properties. CSCs also have been considered as the main cause of cancer recurrence. Targeting CSCs have been suggested as the key for successful treatment against cancer. Tumorsphere cultivation is based on culturing cancer cells onto ultralow attachment surface in serum-free media under the supplementation with growth factors such as epidermal growth factor and basic fibroblast growth factor. Tumorsphere cultivation is widely used to analyze the self-renewal capability of CSCs and to enrich these cells from bulk cancer cells. This method also provides a reliable platform for screening potential anti-CSC agents. The in vitro anti-proliferation activity of potential agents selected from tumorsphere assay is more translatable into in vivo anti-tumorigenic activity compared with general monolayer culture. Tumorsphere assay can also measure the outcome of clinical trials for potential anti-cancer agents. In addition, tumorsphere assay may be a promising strategy in the innovation of future cancer therapeutica and may help in the screening of anti-cancer small-molecule chemicals.

IntroductionHeat 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.MethodsMitogen-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.ResultsHsp27 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.ConclusionsOur 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.