Structure-activity and mechanistic investigations into the cytotoxicity of flavonols in cancer cells

Thesis (MSc)--Stellenbosch University, 2022.ENGLISH ABSTRACT: Flavonols belong to a group of polyphenolic compounds that are found in medicinal and edible plants. They have a broad range of medicinal properties including anti-cancer properties, being able to inhibit cell proliferation and induce apoptosis in cancer cells in the low micromolar range. Previous studies from our lab have found that the flavonol fisetin is cytotoxic to lung cancer cells. The primary aim of this study was to synthesize a library of flavonols, with varying substituents at the 4’- position, and establishing the structure activity relationship (SAR) of the synthesized flavonols. A total of twelve flavonols were synthesized via the Algar -Flyn-Oyamada (AFO) reaction and a thirteenth via the oxidation of the methyl thiol derivative. The cytotoxicity IC50’s of these compounds were determined in MDA-MB-231 and A549 cancer cell lines by making use of a MTT cytotoxicity assay. The flavonols had an overall higher activity towards the A549 cells over MDA-MB-231 cells. The most potent derivatives were 3,7-dihydroxy-2-(4'-trifluoromethylphenyl)-chromen-4-one (34) and 3,7-dihydroxy- 2-(4'-methylsulfonephenyl)-chromen-4-one (36), 8.2 ± 1.8 and 2.9 ± 1.6 μM against A549 cells, respectively. The 4’-substituents had a significant effect on the cytotoxicity of the flavonols and a linear correlation was drawn between the para-Hammett constant of the 4’-substituents and the cytotoxicity. For the flavonols under investigation, the larger the para-Hammett (σp) constant (more electron withdrawing) of the 4’-substituent, the more cytotoxic the flavonols were in A549 cancer cells. Live cell fluorescent imaging was used to determine the subcellular localization of three natural flavonols. A fluorescent tag (dansyl chloride) was incorporated on to a natural flavonol to allow for fluorescent tracking in MDA-MB-231 breast cancer cells. The addition of the dansyl tag to 3,7- dihydroxy-2-phenyl-chromen-4-one (27) was successful and yielded 1-[5- (dimethylamine)naphthalenyl-1-sulfonyl]azetidine-N-{3-[(2-(phenyl)-3-hydroxy-4-oxo-4H-chromen- 7-yl)oxy]propyl (51). Absorption and emission spectra revealed that the flavonols had intrinsic fluorescence and three natural flavonols were used to determine the localization of the flavonols in the MDA-MB-231 cells. Confocal studies revealed that the three natural flavonols had high affinity towards the ER and cell membrane, with less obvious affinity towards the mitochondria. Flavonol 27 was a perfect control to determine the influence of the dansyl tag on 51, the subcellular localization of the dansyl tagged (51) and free (27) flavonol different significantly. Compound 51 had high affinity towards the ER and no other subcellular localization was observed for 51. Dansyl derivatives, lacking pharmacophores, were used as controls and low affinity towards the ER was observed, dansyl chloride did not accumulate in the cells. Two compounds, 7-deazahypoxanthine and a cis-platin derivatives, with known subcellular localization were chosen to be synthesized to be used as positive controls for a fluorescent tag (Dansyl chloride). The synthesis of the precursor 6-[4-(Benzyloxy)benzoyl]-5-phenyl-1H-pyrrolo-[2,3- d]pyrimidine-2,4(3H,7H)-dione (42) was achieved, but unfortunately the synthesis of the 7- deazahypoxanthine derivative remained unsuccessful. The synthesis of the cis-platin (52) derivative, dichloro(ethylenediamine)platinum(II), was successful however the incorporation of the dansyl tag (fluorophore) was not.AFRIKAANSE OPSOMMING: Een van die hoof doelwitte van die studie was om ‘n reeks van flavonole te sintetiseer wat net verskil op die 4’ – posisie. Die reeks was gebruik om die struktuur se invloed op die reaktiwiteit te bepaal deur gebruik te maak van die MTT toetse. Die kragtigste molekules was 3,7-dihydroxy-2-(4'- trifluoromethylphenyl)-chromen-4-one (34) en 3,7-dihydroxy-2-(4'-methylsulfonephenyl)-chromen- 4-one (36), 8.2 ± 1.8 en 2.9 ± 1.6 μM, in die A549 kankersellyn. Die 4’- substituente het ʼn groot inpak op die toksisiteit van die flavonole gehad en ʼn lineêre korrelasie was gevind tussen die para-Hammet konstante en die toksisiteit. Hoe meer elektron onttrekkend die 4’-substituente was, hoe meer aktief was die molekuul. Twee molekules was gekies om gesintetiseer te word omdat hulle bekende sellulêre organel teikens het wat dit toelaat om die molekules te gebruik as ʼn positiewe toets vir die fluorofoor, dansyl kloried. Die molekules was 7-deazahypoxanthine en ʼn cis-platin afleisel. Die sintese van die 7- deazahypoxanthine afleisel was onsuksesvol en die byvoeging van die fluorofoor aan die cis-platin afleisel was onsuksesvol. Die byvoeging van die flurofoor, dansyl kloried, aan 3,7-dihydroxy-2-phenyl- chromen-4-one (27) was suksesvol en die produk, 1-[5-(dimethylamine)naphthalenyl-1- sulfonyl]azetidine-N-{3-[(2-(phenyl)-3-hydroxy-4-oxo-4H-chromen-7-yl)oxy]propyl (51) was gebruik om die invloed van die fluorofoor op die sellulêre opeenhoping van flavonole te bepaal. Absorpsie en emissie spektra het bewys dat die flavonole ʼn natuurlike fluoressensie het en drie is gekies om die sellulêre opeenhoping van die flavonole te bepaal in MDA-MB-231 kankerselle. Soortgelyke studies het getoon dat die flavonole hoë affiniteit het tot sellulêre organe wat groot hoeveelheid membrane besit. Hulle het die grootste affiniteit gehad tot die endoplasmiese retikulum, selmembraan en die mitochondria. Flavonol 27 het in al drie die bogenoemde organe saamgesmelt waar 51 net in die endoplasmiese retikulum saamgesmelt het.Master

A multimedia mobile phone-based youth smoking cessation intervention: findings from content development and piloting studies

BACKGROUND: While most young people who smoke want to quit, few access cessation support services. Mobile phone-based cessation programs are ideal for young people: mobile phones are the most common means of peer communication, and messages can be delivered in an anonymous manner, anywhere, anytime. Following the success of our text messaging smoking cessation program, we developed an innovative multimedia mobile phone smoking cessation intervention. OBJECTIVE: The aim of the study was to develop and pilot test a youth-oriented multimedia smoking cessation intervention delivered solely by mobile phone. METHODS: Development included creating content and building the technology platform. Content development was overseen by an expert group who advised on youth development principles, observational learning (from social cognitive theory), effective smoking cessation interventions, and social marketing. Young people participated in three content development phases (consultation via focus groups and an online survey, content pre-testing, and selection of role models). Video and text messages were then developed, incorporating the findings from this research. Information technology systems were established to support the delivery of the multimedia messages by mobile phone. A pilot study using an abbreviated 4-week program of video and text content tested the reliability of the systems and the acceptability of the intervention. RESULTS: Approximately 180 young people participated in the consultation phase. There was a high priority placed on music for relaxation (75%) and an interest in interacting with others in the program (40% would read messages, 36% would read a blog). Findings from the pre-testing phase (n = 41) included the importance of selecting "real" and "honest" role models with believable stories, and an interest in animations (37%). Of the 15 participants who took part in the pilot study, 13 (87%) were available for follow-up interviews at 4 weeks: 12 participants liked the program or liked it most of the time and found the role model to be believable; 7 liked the role model video messages (5 were unsure); 8 used the extra assistance for cravings; and 9 were happy with two messages per day. Nine participants (60%) stopped smoking during the program. Some technical challenges were encountered during the pilot study. CONCLUSIONS: A multimedia mobile phone smoking cessation program is technically feasible, and the content developed is appropriate for this medium and is acceptable to our target population. These results have informed the design of a 6-month intervention currently being evaluated for its effectiveness in increasing smoking cessation rates in young people

Biological control of postharvest disease in the perishable fruit industry by bacillus lipopeptides

Thesis (MEng)--Stellenbosch University, 2018.ENGLISH SUMMARY: The work presented in this study aimed to investigate the suitability of Bacillus spp. lipopeptides to be used as effective biocontrol agents that have the potential to replace chemically synthesised pesticides that are currently in use in post-harvest disease control. The screening criteria were based on the ability of different Bacillus species to produce antifungal lipopeptides which in turn could inhibit phytopathogens isolated from post-harvest grapes. The study further aimed to identify the lipopeptides responsible for any noted antifungal activity and investigate possible downstream isolation methods that could be included into process optimisation. With these goals in mind, the work presented here hopes to inform on the way forward for Future studies that are focused towards the controlled in vitro production of a customised cell-free biocontrol agent for post-harvest disease control. A phytopathogen culture bank was established from 79 crude isolates of fungi from post-harvest grapes supplied by the South African Table Grape industry. These isolates were purified into 59 pure cultures using 5 sequential rounds of subculturing on PDA, MEA and NA agar plates. Of these, 16 isolates were successfully sequenced using Sanger sequencing with ITS 4 and ITS 5 primers. Of the identified phytopathogens, 50% belonged to Penicillium spp., 33% to Botrytis spp. and the remaining 17% grouped as Aspergillus spp., Alternaria spp. and Lewia spp. These phytopathogens are responsible for noble rot, blue-green mould, bunch rot, black mould and leaf blight in the grape industry respectively. Four different strains of lipopeptide producing bacteria, Bacillus amyloliquefaciens DSM 23117, B. licheniformis DSM 13, B. subtilis ATCC 21332 and B. subtilis spizizini DSM 347 were obtained from culture collections and screened in triplicate in shake flasks over a seven day period using media from Kim et al (1997) with modifications. Bacillus amyloliquefaciens DSM 23117 was identified as the superior producer of lipopeptides. In addition to the significant amounts of lipopeptides produced by this strain, crude lipopeptides from B. amyloliquefaciens were shown to exhibit efficacy (either by causing death or static inhibition) towards all 9 isolated fungal phytopathogen strains tested in radial diffusion assays (RDA). The strains incorporated in RDA assays belonged to Botrytis, Penicillium, Alternaria, Aspergillus and Lewia genera. TLC separation in combination with antifungal efficacy assays further narrowed down the Iturin family, peaks 3, 4, 5, 8 and 9, as compounds that exerted significant antifungal activity. Thus indicating these compounds are important targets for isolation and incorporation into antifungal studies towards the development of cell-free biocontrol products for use in post-harvest applications and crop protection. Using different downstream methods for lipopeptide isolation and purification, salt precipitation was identified as a bulk isolation method while solvent extraction from isobutanol and n-hexane proved superior in separating antifungal lipopeptides from surfactin. Through the use of various analytical and experimental methods all the aims set out in this study were met by large. B. amyloliquefaciens was identified as a target strain for culture and process optimisation, targeting iturins, and for incorporation towards the development of a cell-free biocontrol agent targeting phytopathogens found in, but not limited to, the South African Table Grape Industry.AFRKIKAANSE OPSOMMING: In die studie is die bakterium Bacillus spp bestudeer as moontlike produseerder van anti-fungiese middels wat as plaasvervanger kan dien vir die huidige chemiese middels wat as plaagdoders gebruik word teen fungi. Die kriteria waaraan die siftingstoets onderworpe was, is gebasseer op die vermoeë van die bakterium om anti-fungiese middels te kon produseer en/ of hierdie middels anti-fungiese aktiwiteit kon toon teen die fungi wat ge-isoleer is uit die natuur, meer spesifiek, tafeldruiwe. Met die studie is verder gepoog om die anti-fungiese middels, lipopeptiedes, wat verantwoordelik is vir anti-fungiese aktiwiteite, te identifiseer asook moontlike prosesse te identifiseer vir isolasie. Met die doelwitte word beoog om toekomstige navorsing te lei wat fokus op die in vitro produksie van ‘n selvrye produk wat kan dien as ‘n biologiese plaagdoder. ‘n Kultuurbank vir anti- fungiese eksperimente is geskep vanuit 79 fungiese monsters wat vanaf die Suid-Afrikaanse Tafeldruifbedryf verkry is. Dit is verder omskep in 59 suiwerkulture. DNS identifikasie is gedoen op 18 kulture en 16 is suksesvol geidentifiseer met behulp van DNS herhalings en morfologie studies. Die fungi is gegroep as 50% Penicillium spp., 33% to Botrytis spp. en 17% Aspergillus spp., Alternaria spp. en Lewia spp. saamgegroepeer. Vier spesies van Bacillus naamlik Bacillus amyloliquefaciens DSM 23117, B. licheniformis DSM 13, B. subtilis ATCC 21332 en B. subtilis spizizini DSM 347 is bekom vir die anti-fungiese middels wat hulle produseer, naamlik lipopeptiedes. Bacilluskulture is gekultiveer op selmedia soos gebubliseer deur Kim et al. (1997) met modifikasies en Bacillus amyloliquefaciens is geidentifiseer as die top spesie vir lipopeptiedproduksie. Die resultate van B. amyloliquefaciens gedurende kultivering dui daarop dat die spesie van Bacillus ‘n oorproduseerder is van anti-fungiese middels. Die ongesuiwerde anti-fungiese middels het ook sterk anti-fungiese eienskappe teen al 9 van die fungi-isolate waarteen dit getoets is, getoon. Die fungiese kulture getoets, het behoort aan Botrytis, Penicillium, Alternaria, Aspergillus en Lewia genera. Dunlaag chromatografie in kombinasie met anti-fungiese toetse het Iturins, pieke 3, 4, 5, 8 en 9 geidentifiseer as die middels verantwoordelik vir anti-fungiese aktiwiteit wat die groep ‘n belangrike onderwerp vir verdere studie maak met die doel op die ontwikkeling van ‘n selvrye produk. Deur gebruik te maak van verskillende isolasie- en suiweringsmetodes is soutpresipitasie geidentifiseer as ‘n metode vir algemene isolasie van alle lipopeptiedes terwyl isobutanol en n-hexaan spesifiek anti-fungiese middels geteiken het vir isolasie. Deur die gebruik van verskeie analitiese- en eksperimentele metodes is al die doelwitte wat in hierdie studie uiteengesit is, behaal. B. amyloliquefaciens is geïdentifiseer as 'n teikenspesie vir verdere prosesoptimalisering Iturins en vir inkorporasie in studies vir die ontwikkeling van 'n selvrye, biologiese plaagdoder wat op fytopatogene in die Suid-Afrikaanse Tafeldruifbedryf fokus

Real-time PCR for the detection of Giardia lamblia

Microscopy is considered to be the gold standard for diagnosis of Giardia lamblia infection. However, this method is time-consuming and not very sensitive. We developed a real-time PCR assay based on the small subunit ribosomal RNA gene of G. lamblia for the specific detection of G. lamblia DNA in stool samples and thereafter compared the results with microscopy and antigen detection. The G. lamblia real-time PCR was positive in 102 of 104 fecal samples known to contain G. lamblia cysts and was positive in 10 fecal samples in which G. lamblia antigen was detected but in which no cysts were found with microscopic examination of concentrated fecal samples. The real-time PCR is as specific and sensitive as antigen detection and is more sensitive than microscopy. Moreover, in two patients we were able to detect G. lamblia earlier in the course of infection than with any of the other method

Simultaneous Detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in Fecal Samples by Using Multiplex Real-Time PCR

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples has been considered to be the “gold standard” for diagnosis of E. histolytica, G. lamblia, and C. parvum infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control