ROLES OF LIPOGENESIS IN BREAST CANCER PROGRESSION

Elevated level of lipogenic enzymes and overall lipogenesis have been reported in a wide variety of cancers and blocking the lipogenic pathway by chemical inhibitors or RNA interference causes tumor cell death by apoptosis which provides a strong rationale for targeting lipogenic pathway for the treatment and prevention of cancer however the exact role of lipogenesis as a cause, facilitator or consequence is not yet clearly understood. Therefore in this dissertation research, we set up to determine the mechanism of tumor cell death by inhibiting lipogenesis and to determine the role of increased lipogenesis in the breast cancer progression. In the first part of this study, we investigated the status of fatty acid synthase (FAS) gene which is regarded as the key lipogenic gene in fatty acid biosynthetic pathway and is responsible for the synthesis of lipid molecules by facilitating the condensation reaction between acetyl-CoA and malonyl-CoA in the presence of NADPH. We observed that normal breast epithelial cells MCF10A cells have very low level of FAS expression whereas breast cancer cell lines MCF7, MDA MB231 and MDA MB231 LM have significant overexpression. Next, we observed the similar trend of FAS overexpression in breast cancer stem-like cells (CSCs) isolated from the MCF7, MDA MB231 and MDA MB231 LM cell lines using cell surface markers (CD24-/CD44+/ESA+). These cells were previously transplanted into the mammary fat pad of nude mice and the results of our limiting dilution analysis indicate that CSCs had a significantly higher ability of forming breast cancer in the injected animals which explains our rationale to use CSCs in our research. In order to exploit this lipogenic pathway for the treatment and chemoprevention of breast cancer, we then examined the effects of resveratrol on breast cancer cells. Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. We observed that resveratrol significantly reduced the cell viability by inducing apoptosis in parental cells as well as in CSCs. Resveratrol also inhibited mammosphere formation which is an inherent property of CSCs. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the FAS gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by FAS overexpression suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of CSC in an animal model of human breast cancer xenograft without showing apparental toxicity. Taken together, our results indicate that resveratrol is capable of inducing apoptosis in the CSCs through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol. Taken together, our results indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol. In the second part of research, we tried to determine the role of elevated level of lipogenesis in normal to ductal carcinoma in situ (DCIS) progression. For this, we first analyzed the expression profile of various lipogenic genes using an expression microarray and found that CSCs from DCIS.com showed significantly higher level of ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and FAS than the normal non-tumorigenic stem-like cells obtained from MCF10A. The result was also confirmed by qRT-PCR and Western blot as well as in clinical specimens of DCIS by immunohistochemistry. In the next step, we detected that SREBP1, the master regulator of lipogenic genes, is also upregulated in DCIS and further identified that SREBP1 regulates the co-ordinate expression of ACLY, ACC and FAS ultimately resulting in the elevation of lipogenesis. In order to determine the role of SREBP1 overexpression in normal to DCIS transition, we overexpressed the SREBP1 in MCF10A cells which induced a significant increase in the downstream key lipogenic genes ACLY, ACC1 and FAS which resulted in the clear upregulation of total lipid content in the cells. Furthermore, we found that this elevation of lipogenesis in MCF10A-SREBP1 stem-like cells confers proliferative advantage as well as a significant increase in mammosphere forming ability and anchorage independent growth (3D culture). Thus, our results showed a possibility that increased lipogenesis in normal stem-like cells may be responsible for providing oncogenic transformation properties which can be confirmed at least in our in vitro model. We then examined the effects of resveratrol on CSCs sorted from DCIS.com. We found that resveratrol decreased the cell viability and increased apoptosis by reducing the total lipid content by inhibiting the expression of SREBP1 and downstream lipogenic genes. Resveratrol also hindered the stemness of the DCIS CSCs by inhibiting its mammosphere forming ability. When DCIS CSCs were transplanted into mammary fat pad of nude mice which were on resveratrol treatment, we observed that resveratrol significantly suppressed the formation of DCIS by downregulating lipogenic genes and by upregulating pro-apoptotic genes, DAPK2 and BNIP3. Collectively, our results indicate that lipogenic genes SREBP1 co-ordinately regulates the overexpression of ACLY, ACC1 and FAS in DCIS CSCs at an early stage of breast tumorigenesis and thus confer proliferative and survival advantages. Anti-growth effect of resveratrol on DCIS CSCs also provides us with a strong rationale to use this agent for chemo-prevention against DCIS

Adsorptive Removal of Methyl Red from Aqueous Solution onto Charred and Aminated Sugarcane Waste

Abstract: Adsorptive removal of Methyl Red (MR) from aqueous solution onto chemically modified charred sugarcane waste (CSW) and aminated sugarcane waste (ASW) has been investigated. The surface modification was characterized by FTIR, SEM, elemental analysis and Boehm titration. The effect of pH, contact time and MR concentrations were studied by batch equilibrium method. Maximum dye removal was observed at pH 2 onto CSW while that for ASW at pH 7. The dye can be quantitatively removed onto the surface of these adsorbents at a contact time of 3 h. Maximum adsorption capacity (qmax) for the CSW and ASW were found to be 125.0 mg/g and 142.85 mg/g, respectively. Adsorption kinetic data were tested using pseudo-first order, pseudo-second order and intra-particle diffusion models. Kinetic studies revealed that the adsorptive removal of the dye onto the adsorbents followed pseudo-second order kinetics model. The obtained results indicated an excellent alternative for the treatment of dye contaminated wastewater using such chemically modified sugarcane waste at low cost with better efficiency

The burden of injuries in Nepal: Findings from the NIHR Global Health Research Group

Background: Injuries cause significant harm and may lead to disability yet are largely preventable. Understanding the epidemiology and determinants of injury in any given context is an essential step towards effective prevention. In Nepal, surveys suggest that injuries on the road, at home and at work are a problem, but in the absence of injury surveillance, robust death registration or police records, the true burden is unclear. For those who are injured, access to prehospital care is variable. Objectives: (i) To understand the epidemiology of injuries (ii) To identify potentially modifiable risk factors to inform the development of prevention interventions (iii) To build capacity and capability for injury prevention research.Design: Observational, secondary data analysis and qualitative methods were used. We worked with communities, practitioners and stakeholders to identify potential participants, develop study protocols and disseminate findings.Setting: Nepal.Participants: Patients, communities and road users, health system practitioners and managers, professionals (e.g. police, engineers, journalists) and local and national decision makers.Main outcome measures: epidemiological evidence of the burden of injuries, evidence to inform future intervention development. Data sources: participants, health services, police and information in the public domain.Review methods: Reviews were conducted systematically with evidence synthesised narratively.Results: The Nepal Injury Research Centre was established, and a cadre of researchers trained. Three researchers and our data manager completed Masters degree courses and all researchers developed their skills by leading at least one project from protocol development through to publication. A review of publications reporting injuries indicated that existing epidemiological evidence mostly arose from case series at high risk of bias. A review of existing legislation showed policy gaps and incomplete implementation or enforcement. Surveillance studies and a household survey showed the high burden of injuries at home, work and on the roads and the neglected issue of suicide. Previously unreported inequalities by age, sex, ethnic group and income level were identified. Existing health, police and death registration data systems are at high risk of under-reporting and misclassification. Road traffic injury emerged as a major concern; road users fear being injured as pedestrians, passengers or drivers, the economic burden of road injuries has increased three-fold over eight years and potentially modifiable risk factors were identified. The provision of first response services is highly variable, and the public and practitioners are fearful of prosecution in the event of poor outcomes. We found it is feasible to train the traffic police in first response and for them to use their skills at traffic collisions. Research priorities for suicide prevention were identified.Limitations: Studies were limited by the quality of the data available through existing systems, with data often incomplete or poorly coded. Our studies were largely conducted in one district with topography typical of many areas of Nepal. However, our findings may not be generalisable to all districts. Conclusions: Our programme identified the inequitable and significant burden of injuries in Nepal. There is the potential to develop existing legislation and health and transport systems to reduce the incidence and consequences of injury.Future work: Research should focus on interventions to reduce injury risk on the roads and at home/work, to develop the first response system and standardise care, and to strengthen injury data systems