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Does degradation of human vault RNA3 by RNA interference reduce multidrug resistance in GLC4/REV, a small-cell lung cancer cell line?Adam, Michael R. January 2004 (has links)
Vaults, recently discovered in 1986, are multi-subunit organelles with a molecular mass of ,--,13 MDa. The specific function of vaults is unknown, although they are believed to be involved in internal transport. These ribonucleoproteins are composed of the major vault protein, which comprises ' 70% of the vault's mass, two minor proteins, TEP1 and vPARP, and untranslated RNA(s). It is believed that the protein components of the vault are structural while the RNAs are the functional components. Implications of the vault's involvement in multi-drug resistance in cancer have been made. In some resistant cancer cells, the major vault protein and vRNA(s) are up-regulated up to 15 times when cells are exposed to a cytotoxic drug. Cytotoxic drugs such as doxorubicin are administered as a cancer treatment, but may be ineffective because the drug is actively pumped out of the cell. Multi-drug resistance is the most common failure of chemotherapeutic cancer treatment. In order to prevent the development of multi-drug resistance this research employed the use of small interfering RNA technology to down-regulate the expression of one of the vault RNAs, vRNA3, in cultured GLC4 cells, a small-cell lung cancer cell line. If the vRNA(s) are the functional portion of the vault and a cloned siRNA prevents their up-regulation after drug exposure, the cells should lose their multi-drug resistance, stimulating apoptosis. If successful, this approach may provide an alternative approach to cancer treatment in cells which respond to chemotherapy by increasing the number of vault particles.Initially, the transfection of a plasmid into GLC4 cells was optimized. The best transfection efficiency (N20%) was obtained by using GeneTherapySystems' GenePORTER2 transfection reagent in serum free conditions. To determine if the vault RNAs are the functional portion of the vault complex that confers multi-drug resistance to a cell, a small interfering RNA fragment was designed to specifically knock-down the expression of human vault RNA 3. The siRNA sequence homologous to a portion of vault RNA3 was cloned into an expression vector, and using optimized transfection protocols was transfected into GLC4/REV cells. A Western analysis using caspase-8 antibodies showed no difference in caspase-8 expression in doxorubicin treated and untreated cells. Preliminary results yielded by reverse transcriptase polymerase chain reaction amplification of isolated RNA indicated that the vRNAs were not down-regulated by the siRNAs. / Department of Biology
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Determining the anti-cancer properties of zinc and novel quinoxaline derivatives on lung cancer cellsSibiya, Mixo Aunny January 2020 (has links)
Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2020 / Despite major advancements in the development of various chemotherapuetic agents,
treatment for lung cancer remains costly, ineffective, toxic to neighbouring normal noncancerous cells and still hampered by high level of remissions (Wistuba et al., 2018;
Tana et al., 2016; Schiller et al., 2002). Synthesis of novel quinoxalines with a wide
spectrum of biological activities has recently received considerable attention with
promising anticancer drug activity since most of them do not affect non-cancerous
cells and are derived from readily available less costly raw materials (Srivastava et al.,
2014). Since combination treatment has been shown to augment and improve single
drug treatment, trace elements were employed in this study in combination with
quinoxalines derivatives (Gomez et al., 2016; Kocdor et al., 2015; Ku et al., 2012; John
et al., 2010; Killile and Killilea, 2007). Zinc is an essential element that is integral to
many proteins and transcription factors which regulate key cellular functions such as
the response to oxidative stress, DNA replication, DNA damage repair, cell cycle
progression, and apoptosis (Dhawan and Chadha, 2010). Owing to the importance of
these two approaches, the aim of this study was to provide in vitro preliminary
anticancer activity data on A549 lung cancer cells using combination of zinc and
quinoxaline derivatives. An assessment of the quinoxaline derivatives ferric reducing
power and DPPH free radical scavenging activity was performed. The cytotoxic and
anti-proliferation activity of these derivatives and zinc on cancer cell lines was
determined using the MTT assay. The ability of the quinoxaline derivatives and zinc to
modulate oxidative stress was evaluated using the H2DCFDA fluorescence assay. Cell
cycle arrest stages were analysed by flow cytometry through propidium iodide cell
cycle analyses. The ability of the quinoxaline derivatives to induce apoptosis in cancer
cells was assessed using DAPI/PI, Acridine Orange/Ethidium Bromide (AO/EB) and
Annexin V-FITC/Dead Cell assays. Western blot was used to investigate the Bcl/Bax
expression ratios in A549 lung cancer cells after treatment with quinoxaline
derivatives, zinc and in combination. Of the four quinoxaline derivatives tested, 3-(quinoxaline-3-yl) prop-2-ynyl methanosulphate (LA-39B) and 3-(quinoxaline-3-yl) prop-2-yn-1-ol (LA-55) produced significant anticancer properties against A549 lung cancer cells at minimal concentrations of 25μM. Both quinoxaline derivatives displayed antioxidant properties and did not induce cell death in non-cancerous Raw 267.4 macrophage cells.
Cytotoxicity was observed in A549 lung cancer, HeLa cervical cancer and MCF-7
breast cancer cells albeit inhibition was more pronounced in A549 lung cancer cells.
Treatment of cancer cells with zinc also resulted in pronounced cytotoxicity at a
minimal concentration of 25μM. Although reduced oxidative stress was observed in
Raw 264.7 macrophages, in A549 lung cancer cells both compounds were able to
increase ROS production which was accompanied by high levels of apoptosis when
treated with derivatives and zinc alone but when in combination an improved higher
level of apoptosis is observed. The improved anti-cancer activity of this drug
combination treatment was further accompanied by lower Bcl/Bax expression ratios
with upregulation of Bax in A549 lung cancer cells. The results of the study suggest
that 3-(quinoxaline-3-yl) prop-2-ynyl methanosulphate and 3-(quinoxaline-3-yl) prop-
2-yn-1-ol are potential candidates drug for treatment of lung cancer. The use of these
quinoxaline derivatives in combination with zinc can offer alternative treatment options
for lung cancer. / National Research Foundation (NRF)
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