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High glucose (HG), a hallmark of the tumour microenvironment, is also a biomechanical stressor, as it exerts hyper-osmotic stress (HG-HO), but not much is known regarding how tumour cells mechanoadapt to HG-HO. Therefore, this study aimed to delineate the novel molecular mechanisms by which tumour cells mechanoadapt to HG/HG-HO and whether phytochemical-based interference in these mechanisms can generate tumour-cell-selective vulnerability to cell death. Mannitol and L-glucose were used as hyper-osmotic equivalents of high glucose. The results revealed that the tumour cells can efficiently mechanoadapt to HG-HO only in the normoxic microenvironment. Under normoxic HG/HG-HO stress, tumour cells polySUMOylate a higher pool of mitotic driver pH3(Ser10), which translocates to the nucleus and promotes faster cell divisions. On the contrary, acute hypoxia dampens HG/HG-HO-associated excessive proliferation by upregulating sentrin protease SENP7. SENP7 promotes abnormal SUMOylation of pH3(Ser10), thereby restricting its nuclear entry and promoting the M-phase arrest and cell loss. However, the hypoxia-arrested cells that managed to survive showed relapse upon reversal to normoxia as well as upregulation of pro-survival-associated SENP1, and players in tumour growth signalling, autophagy, glycolytic pathways etc. Depletion of SENP1 in both normoxia and hypoxia caused significant loss of tumour cells vs undepleted controls. SENP1 was ascertained to restrict the abnormal SUMOylation of pH3(Ser10) in both normoxia and hypoxia, although not so efficiently in hypoxia, due to the opposing activity of SENP7. Co-treatment with Momordin Ic (MC), a natural SENP1 inhibitor, and Gallic Acid (GA), an inhibitor of identified major pro-tumourigenic signalling (both enriched in Momordica charantia), eliminated surviving tumour cells in normal glucose, HG and HG-HO normoxic and hypoxic microenvironments, suggesting that appropriate and enhanced polySUMOylation of pH3(Ser10) in response to HG/HG-HO stress was attenuated by this treatment along with further dampening of other key tumourigenic signalling, due to which tumour cells could no longer proliferate and grow.

Shasha Zhao, Xinyuan Gao, Chunlei Zhong, Yunxia Li, Ming Wang, Shuzhi Zang Department of Respiratory Medicine, The First Hospital Affiliated to the Xinxiang Medical College, Weihui, Henan 453100, People’s Republic of ChinaCorrespondence: Shasha Zhao Department of Respiratory MedicineThe First Hospital Affiliated to the Xinxiang Medical College, No. 88 Jiankang Road, Weihui, Henan 453100, People’s Republic of ChinaTel +86-0373-4403114Email zhaossxxyxy06@163.comBackground: Tumour growth and development are dependent on many factors including long noncoding RNAs (lncRNAs). However, limited information is available on the involvement of lncRNAs in non-small cell lung cancer (NSCLC) and the molecular mechanisms have not been defined. Here, we examined the expression of small nucleolar RNA host gene 3 (SNHG3) and its contribution to the development of NSCLC.Methods: We detected SNHG3, miR-216a, and ZEB1 expression in tissues from NSCLC patients and lung adenocarcinoma cell lines using quantitative real-time polymerase chain reaction. Proliferation, migrations, invasion, and apoptosis of tumour cells were assessed using cell counting kit-8, transwell experiments, and flow cytometry after SNHG3 knockdown by small interfering RNAs. Bioinformatics and luciferase reporter assays were employed for analysing the interactions between SNHG3, miR-216a, and ZEB1.Results: We found highly upregulated SNHG3 in tissues and cells from NSCLC patients, which was linked to poor prognosis. SNHG3 silencing diminished the ability of NSCLC cells to proliferate, migrate, and invade and promoted apoptosis. Furthermore, SNHG3 competed with endogenous RNA and enhanced the expression of ZEB1 by interfering with miR-216a. ZEB1 overexpression or miR-216a blockade reversed SNHG3-induced tumour inhibition. Similar effects were observed in vivo where SNHG3 knockdown inhibited NSCLC tumour growth by reducing expression of miR-216a while increasing that of ZEB1.Conclusion: Knockdown of SNHG3 inhibits NSCLC tumour development and progression by upregulation of ZEB1 and interference with miR-216a, revealing an attractive alternative target for patients with NSCLC.Keywords: lncRNA SNHG3, competing endogenous RNA, NSCLC, tumour proliferation, tumour migration, tumour invasion, tumour apoptosis

Shasha Zhao, Xinyuan Gao, Chunlei Zhong, Yunxia Li, Ming Wang, Shuzhi Zang Department of Respiratory Medicine, The First Hospital Affiliated to the Xinxiang Medical College, Weihui, Henan 453100, People’s Republic of ChinaCorrespondence: Shasha Zhao Department of Respiratory MedicineThe First Hospital Affiliated to the Xinxiang Medical College, No. 88 Jiankang Road, Weihui, Henan 453100, People’s Republic of ChinaTel +86-0373-4403114Email zhaossxxyxy06@163.comBackground: Tumour growth and development are dependent on many factors including long noncoding RNAs (lncRNAs). However, limited information is available on the involvement of lncRNAs in non-small cell lung cancer (NSCLC) and the molecular mechanisms have not been defined. Here, we examined the expression of small nucleolar RNA host gene 3 (SNHG3) and its contribution to the development of NSCLC.Methods: We detected SNHG3, miR-216a, and ZEB1 expression in tissues from NSCLC patients and lung adenocarcinoma cell lines using quantitative real-time polymerase chain reaction. Proliferation, migrations, invasion, and apoptosis of tumour cells were assessed using cell counting kit-8, transwell experiments, and flow cytometry after SNHG3 knockdown by small interfering RNAs. Bioinformatics and luciferase reporter assays were employed for analysing the interactions between SNHG3, miR-216a, and ZEB1.Results: We found highly upregulated SNHG3 in tissues and cells from NSCLC patients, which was linked to poor prognosis. SNHG3 silencing diminished the ability of NSCLC cells to proliferate, migrate, and invade and promoted apoptosis. Furthermore, SNHG3 competed with endogenous RNA and enhanced the expression of ZEB1 by interfering with miR-216a. ZEB1 overexpression or miR-216a blockade reversed SNHG3-induced tumour inhibition. Similar effects were observed in vivo where SNHG3 knockdown inhibited NSCLC tumour growth by reducing expression of miR-216a while increasing that of ZEB1.Conclusion: Knockdown of SNHG3 inhibits NSCLC tumour development and progression by upregulation of ZEB1 and interference with miR-216a, revealing an attractive alternative target for patients with NSCLC.Keywords: lncRNA SNHG3, competing endogenous RNA, NSCLC, tumour proliferation, tumour migration, tumour invasion, tumour apoptosis

Background and aims Black tea and green tea were produced via different processing techniques from the same tea leave variety. Then, biochemical components of the two water extracts were analysed to study cell apoptosis, migration and invasion of HepG2 cells induced by black tea and green tea. Method The monomer components of the black tea and green tea extracts were analysed by colorimetry and HPLC, with MTT assay and colony formation assays used to assess cell proliferation and viability. The effects of black tea and green tea on apoptosis of HepG2 cells were verified by flow cytometry, with wound healing and Transwell experiments used to detect cell invasion and metastasis. The expression of PI3K/Akt signalling and apoptosis-related proteins as well as epithelial-mesenchymal transition (EMT) regulatory factor in HepG2 cells were determined by western blotting after black tea and green tea treatment. Results and conclusions Black tea and green tea extracts demonstrated different degrees of inhibition of cell migration and invasion, with green tea inducing more HepG2 cell apoptosis. In addition, green tea and black tea extracts inhibited the growth of HepG2 cells and induced apoptosis via PI3K/Akt, and inhibited cell migration and invasion through the MMPs signalling pathway. This study revealed the effects of fermented (black tea) and non-fermented tea (green tea) on liver cancer cells, providing a basis for the investigation of tea extracts for their anti-tumour potential.

We evaluated the pre-clinical efficacy of a novel intraperitoneal (i.p.) sustained-release paclitaxel formulation (PTX(ePC)) using bioluminescent imaging (BLI) in the treatment of ovarian cancer. Human ovarian carcinoma cells stably expressing the firefly luciferase gene (SKOV3(Luc)) were injected i.p. into SCID mice. Tumour growth was evaluated during sustained or intermittent courses of i.p. treatment with paclitaxel (PTX). In vitro bioluminescence strongly correlated with cell survival and cytotoxicity. Bioluminescent imaging detected tumours before their macroscopic appearance and strongly correlated with tumour weight and survival. As compared with intermittent therapy with Taxol, sustained PTX(ePC) therapy resulted in significant reduction of tumour proliferation, weight and BLI signal intensity, enhanced apoptosis and increased survival times. Our results demonstrate that BLI is a useful tool in the pre-clinical evaluation of therapeutic interventions for ovarian cancer. Moreover, these results provide evidence of enhanced therapeutic efficacy with the sustained PTX(ePC) implant system, which could potentially translate into successful clinical outcomes.

The breast cancer diagnosis covers a wide range of tumours with different geno- and phenotypic characteristics. The diversity among studies on life-style factors and their impact on breast cancer risk might be clarified by recognizing breast cancer as a heterogeneous disease. A major aim of this thesis was to investigate the association between several life-style factors and subsequent breast cancer risk according to pathological- and tumour biological characteristics. Further, we examined two novel tumour markers with respect to their relation to established tumour characteristics, estrogen-related-life-style-factors, and/or response to endocrine breast cancer treatment. The thesis is predominantly based on breast cancer cases from the Malmö Diet and Cancer Study, a prospective cohort study including 17 035 women with baseline examinations performed 1991-1996. The study cohort was followed by record-linkage with national cancer registries and at the end of follow-up in 2004, 622 women had been diagnosed with breast cancer. Additionally, we used a consecutive cohort of 512 women diagnosed with breast cancer at the Malmö University Hospital 1988 and 1992. Immunohistochemical assessments of different tumour markers were performed using the tissue microarray (TMA) technique. Analyses of dietary habits and tumour characteristics revealed an association between low energy intakes and a more aggressive histopathological phenotype as well as over-expression of the cell cycle regulating protein, cyclin D1. A low fat intake showed similar associations to tumour characteristics. The use of combined (estrogen-progestin) hormone replacement therapy was associated with an increased risk of breast cancer, although with comparatively favourable prognostics factors, such as lobular type, low grade, low proliferation, negative/low cyclin D1 expression and maintained expression of the tumour suppressor gene, p27. In a similar way obesity was associated with an increased risk of less aggressive breast cancer characterised by lobular type, grade II, low proliferation, HER2 negativity, ERα+, PgR+, but negative ERβ expression. The enzyme HMG-CoA reductase was differentially expressed in breast cancer and high expression was associated with a smaller tumour size, low grade, low proliferation, and expression of ERα and ERβ, but not PgR. Estrogen-related factors, such as obesity and use of hormonal replacement, were associated with an increased risk of tumours with high expression of HMG-CoA reductase. The novel estrogen receptor, ERβ, was associated with expression of ERα and PgR, but not with other established prognostic factors, such as tumour size, grade, and lymph node metastases. Co-expression of both estrogen receptors indicated an improved response to endocrine therapy.

Multi-drug resistance (MDR) to chemotherapy is the major challenge in the treatment of cancer. MDR can develop by numerous mechanisms including decreased drug uptake, increased drug efflux and the failure to undergo drug-induced apoptosis. Evasion of drug-induced apoptosis through modulation of ion transporters is the main focus of this paper and we demonstrate how pro-apoptotic ion channels are downregulated, while anti-apoptotic ion transporters are upregulated in MDR. We also discuss whether upregulation of ion transport proteins that are important for proliferation contribute to MDR. Finally, we discuss the possibility that the development of MDR involves sequential and localized upregulation of ion channels involved in proliferation and migration and a concomitant global and persistent downregulation of ion channels involved in apoptosis.

Background The incidence of Cholangiocellular Carcinoma (CCA) is increasing in the western world. The tumour has a high proportion of desmoplastic stroma and is correlated with a worse prognosis when cancer associated myofibroblasts (CAFs) are present. Recent studies showed promising results after liver transplantation (LTx) in non-resectable early stage CCA. Mycophenolic acid (MPA) and the mTor inhibitor Everolimus are used to prevent organ rejection but recently were shown to exhibit an antiproliferative effect on CCA-cells. Little is known about the influence of immunosuppressive drugs on tumour cell proliferation and migration after paracrine stimulation by CAFs. Moreover, it is still unknown, which signaling pathways are activated following these specific cell-cell interactions. Methods CCA cell lines HuCCT1 and TFK1 were utilized for the study. CAFs were derived from resected CCA cancer tissue. Cell viability was measured by the crystal violet assay and tumour cell invasion was quantified using a modified co-culture transmigration assay. Semiquantitative cytokine-expression was measured using a cytokine-array. Protein expression and phosphorylation of ERK, STAT3 and AKT was determined by Western-blot analysis. Results CCA cells treated with MPA exhibited a dose related decrease in cell viability in contrast to Cyclosporine A (CSA) treatment which had no effect on cell viability. Everolimus significantly inhibited proliferation at very low concentrations. The pro-invasive effect of CAFs in co-culture transmigration assay was significantly reduced by Everolimus at a concentration of 1nM (p = 0.047). In contrast, MPA and CSA showed no effect on tumour cell invasion. Treatment of CAFs with 1nM Everolimus showed a significant reduction in the expression of IL 8, IL 13, MCP1, MIF and Serpin E1. CCA-cells showed significant increases in phosphorylation of ERK, STAT3 and AKT under the influence of conditioned CAF-media. This effect was suppressed by Everolimus. Conclusions The secretion of proinflammatory cytokines by CAFs may lead to increased activation of JAK/STAT3-, ERK- and AKT-signaling and increased migration of CCA-cells. Everolimus abrogates this effect and inhibits proliferation of CCA-cells even at low concentrations. LTx for non-resectable early stage CCA is currently performed in several clinical studies. Consistent with a role for common immunosuppressants in inhibiting tumour cell-proliferation and -invasion, our study indicates that a combination of standard therapies with Everolimus and MPA is a promising therapy option to treat CCA following LTx.

Background Quantification of kinetic parameters of positron emission tomography (PET) imaging agents normally requires collecting arterial blood samples which is inconvenient for patients and difficult to implement in routine clinical practice. The aim of this study was to investigate whether a population-based input function (POP-IF) reliant on only a few individual discrete samples allows accurate estimates of tumour proliferation using [18F]fluorothymidine (FLT). Methods Thirty-six historical FLT-PET data with concurrent arterial sampling were available for this study. A population average of baseline scans blood data was constructed using leave-one-out cross-validation for each scan and used in conjunction with individual blood samples. Three limited sampling protocols were investigated including, respectively, only seven (POP-IF7), five (POP-IF5) and three (POP-IF3) discrete samples of the historical dataset. Additionally, using the three-point protocol, we derived a POP-IF3M, the only input function which was not corrected for the fraction of radiolabelled metabolites present in blood. The kinetic parameter for net FLT retention at steady state, Ki, was derived using the modified Patlak plot and compared with the original full arterial set for validation. Results Small percentage differences in the area under the curve between all the POP-IFs and full arterial sampling IF was found over 60 min (4.2%-5.7%), while there were, as expected, larger differences in the peak position and peak height. A high correlation between Ki values calculated using the original arterial input function and all the population-derived IFs was observed (R2 = 0.85-0.98). The population-based input showed good intra-subject reproducibility of Ki values (R2 = 0.81-0.94) and good correlation (R2 = 0.60-0.85) with Ki-67. Conclusions Input functions generated using these simplified protocols over scan duration of 60 min estimate net PET-FLT retention with reasonable accuracy.