<p>Double immunoflourecence was done to determine the compounds' effect after 24 h on m... more <p>Double immunoflourecence was done to determine the compounds' effect after 24 h on microtubule dynamics within MDA-MB-231 cells. Tyrosinated (dynamic) microtubules are visualized as red, whereas the detyrosinated microtubules are stained green. The vehicle-treated cells (A) demonstrated an intact dynamic microtubule structure. The colchicine-exposed positive control cells (B) showed complete microtubule depolymerisation with few detyrosinated microtubule fragments remaining. ESE-16 (C), ESE-15-one (D) and EMBS-treated cells (E) demonstrated detyrosinated microtubule fragments. All treated cell samples demonstrated a decrease in cell density with rounded, shrunken cells. Apoptotic bodies are evident in the ESE-16 treated cells (X63 oil objective).</p
<p>LDH levels of ESE-15-one, EMBS and ESE-16 -treated HeLa cells and vehicle-treated contro... more <p>LDH levels of ESE-15-one, EMBS and ESE-16 -treated HeLa cells and vehicle-treated control cells after 24 h exposure. The background control consists of growth medium only. The low control refers to cells resuspended in growth medium and the high control to cells resuspended in growth medium with cell lysis solution added to the cells shortly before the experiment was terminated (according to the manufacturer's instructions). No statistically significant increase in LDH levels was observed in treated cells. These results showed that the sulphamoylated compounds were not toxic to the cells.</p
<p>Flow cytometry was conducted to present the effects of these sulphamoylated compounds on... more <p>Flow cytometry was conducted to present the effects of these sulphamoylated compounds on cytochrome <i>c</i> release from the mitochondria into the cytoplasm. Vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate-treated cells (B), ESE-16-treated cells (C), ESE-15-one-treated cells (D) and EMBS-treated cells (E) following 24 h of exposure. All three compound-treated cells demonstrated increased levels of cytochrome <i>c</i> release when compared to the vehicle-treated cells.</p
<p>Caspase 8 and caspase 6 activity ratios of compound- and actinomycin D-treated cells com... more <p>Caspase 8 and caspase 6 activity ratios of compound- and actinomycin D-treated cells compared to vehicle-treated cells. Caspase 6 and caspase 8 activities in all compound-treated cells increased when compared to vehicle-treated cells. EMBS-treated cells demonstrated the most prominent increase in caspase 8 activity and ESE-16-treated cells the most prominent increase in caspase 6 activity when compared to vehicle-treated cells. 2-Methoxyestradiol-bis-sulphamate-treated cells are illustrated in the figure as 2MEBM due to limited space. An asterisk (*) indicates a statistically significant <i>P</i>-value of <0.05 when compared to vehicle-treated cells.</p
<p>HeLa cell numbers expressed as a % of cells relative to the control (cells propagated in... more <p>HeLa cell numbers expressed as a % of cells relative to the control (cells propagated in growth medium) after exposure to ESE-15-one, EMBS and ESE-16 for 24 h. An asterisk (*) indicates a statistically significant <i>P</i>-value of <0.05 when compared to cells propagated in growth medium.</p
<p>TEM revealed vehicle-treated cells displaying no signs of distress (A). Cells treated wi... more <p>TEM revealed vehicle-treated cells displaying no signs of distress (A). Cells treated with 2-methoxyestradiol-bis-sulphamate (B), ESE-16 (C) and ESE-15-one (D) demonstrated the occurrence of apoptotic bodies and vacuole formation. Cells treated with EMBS (E) displayed an increase in the number of vacuoles.</p
<p>Spectrophotometry results of caspase 3 activity indicated that after exposure to these s... more <p>Spectrophotometry results of caspase 3 activity indicated that after exposure to these sulphamoylated compounds caspase 3 activity increased significantly. An asterisk (*) indicates a statistically significant <i>P</i>-value <0.05 when compared to cells propagated in growth medium.</p
<p>Tubulin was incubated with the compounds ((A), ESE-16 (B), ESE-15-one (C) and EMBS (D)),... more <p>Tubulin was incubated with the compounds ((A), ESE-16 (B), ESE-15-one (C) and EMBS (D)), as described in the methods section. This assay showed that ESE-16 had a pronounced effect comparable with that of colchicine.</p
<p>Mitotracker-stained vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate... more <p>Mitotracker-stained vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate-treated cells (B), ESE-16-treated cells (C), ESE-15-one-treated cells (D) and EMBS-treated cells (E) after 24 h exposure. An increase in the number of cells with reduced mitochondrial potential treated with 2ME2 analogues compared to the vehicle-treated control cells was observed.</p
<p>Double immunoflourecence was conducted to determine the compounds' effect after 24 h... more <p>Double immunoflourecence was conducted to determine the compounds' effect after 24 h on microtubule dynamics within HeLa cells. Tyrosinated (dynamic) microtubules are visualized as red, whereas the detyrosinated (stable or stabilized) microtubules are stained in green. The vehicle-treated cells (A) demonstrated an intact dynamic microtubule structure. Both the colchicine positive control (B), as well as the ESE-16-treated cells (C) showed complete microtubule depolymerisation with few detyrosinated microtubule fragments remaining. ESE-15-one-treated cells (D) demonstrated altered microtubule morphology. EMBS-exposed cells (E) revealed fragmented microtubules. All treated cell samples demonstrated a decrease in cell density and rounded cells (X63 oil objective).</p
<p>Mitocapture-stained vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate... more <p>Mitocapture-stained vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate-treated cells (B), ESE-16-treated cells (C), ESE-15-one-treated cells (D) and EMBS-treated cells (E) after 24 h exposure. An increase in the number of cells presenting with compromised mitochondrial potential was demonstrated in all three compound-treated cells when compared to the vehicle-treated control cells.</p
<p>Flow cytometry was used to show the effects of these compounds on cytochrome <i>c&... more <p>Flow cytometry was used to show the effects of these compounds on cytochrome <i>c</i> release from the mitochondria into the cytoplasm in HeLa cells. Vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate-treated cells (B), ESE-16-treated cells (C), ESE-15-one-treated cells (D) and EMBS-treated cells (E) following 24 h of exposure. Cytochrome <i>c</i> release was evident in all three compound-treated cells when compared to the vehicle-treated cells.</p
2-Methoxyestradiol (2ME2) is a natural metabolite of 17-β-estradiol exerting both antiproliferati... more 2-Methoxyestradiol (2ME2) is a natural metabolite of 17-β-estradiol exerting both antiproliferative and antiangiogenic characteristics. Due to limited bioavailability, analogues of 2ME2 were designed, synthesized and evaluated for improved in vitro antiproliferative activity as well as bioavailability. 2-Methoxyestradiol-bis-sulphamate (2-MeOE2bisMATE) is an analogue of 2ME2 which was produced by sulphamoylation of the two hydroxyl groups on carbons 3 and 17. The aim of this in vitro study was to evaluate the influence of 2MeOE2bisMATE on morphology, apoptosis and autophagy induction in an oesophageal carcinoma (SNO) cell line by means of transmission electron microscopy (TEM) and flow cytometry (cyto-ID and LC3 autophagy detection assays). In 2-MeOE2bisMATE-treated cells, features of both apoptosis and autophagy such as the presence of apoptotic bodies and autophagy vesicles were observed using the transmission electron microscopy. A significant increase in the quantity of autophag...
Current chemotherapeutic agents have many side effects and are toxic to normal cells, providing i... more Current chemotherapeutic agents have many side effects and are toxic to normal cells, providing impetus to identify agents that can effectively eliminate tumorigenic cells without damaging healthy cells. The aim of this study was to examine whether combining a novel BRD4 inhibitor, ITH-47, with the antimitotic estradiol analogue, ESE-15-ol, would have a synergistic effect on inhibiting the growth of two different breast cancer cell lines in vitro. Our docking and molecular dynamics studies showed that compared to JQ1, ITH-47 showed a similar binding mode with hydrogen bonds forming between the ligand nitrogens of the pyrazole, ASN99, and water of the BRD4 protein. Data from cell growth studies revealed that the GI50 of ITH-47 and ESE-15-ol after 48 hours of exposure was determined to be 15 μM and 70 nM, respectively, in metastatic MDA-MB-231 breast cancer cells. In tumorigenic MCF-7 breast cancer cells, the GI50 of ITH-47 and ESE-15-ol was 75 μM and 60 nM, respectively, after 48 hou...
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2012
In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel ... more In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17-beta-estradiol analog in mcf-7 breast adenocarcinoma cells. After a 24 hour exposure time, cells both apoptosis and autophagy were induced.
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2013
In vitro evaluering van 2-metoksieestradiol-bis-sulfamaat op selgroei, morfologie, selsiklus en d... more In vitro evaluering van 2-metoksieestradiol-bis-sulfamaat op selgroei, morfologie, selsiklus en die moontlike induksie van tipe seldood in 'n esofagus karsinoom senyn', Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie 32(1), Art.
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2014
2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19) induces cell death via the autophagy in b... more 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19) induces cell death via the autophagy in breast adenocarcinoma cells. C19 induces cell death via autophagy in breast adenocarcinoma cells by increasing the number of acidic vacuoles formed and an increase in ROS generation, as well as accumulation of autophagosomes.
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2012
Using a novel synthesised sulphamoylated 2-methoxyestradiol analogue, C19, two types of cell deat... more Using a novel synthesised sulphamoylated 2-methoxyestradiol analogue, C19, two types of cell death, namely apoptosis and autophagy, were demonstrated in vitro when cervical cancer HeLa cells were exposed to this compound.
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2014
Effects of estradiol analogues on blood: A pilot study. This study demonstrated that both ESE-15-... more Effects of estradiol analogues on blood: A pilot study. This study demonstrated that both ESE-15-ol en ESE-16 have no effect on blood cells. Future ex vivo and in vivo studies into the mechanism of these potentially anticancer drugs are warranted.
<p>Double immunoflourecence was done to determine the compounds' effect after 24 h on m... more <p>Double immunoflourecence was done to determine the compounds' effect after 24 h on microtubule dynamics within MDA-MB-231 cells. Tyrosinated (dynamic) microtubules are visualized as red, whereas the detyrosinated microtubules are stained green. The vehicle-treated cells (A) demonstrated an intact dynamic microtubule structure. The colchicine-exposed positive control cells (B) showed complete microtubule depolymerisation with few detyrosinated microtubule fragments remaining. ESE-16 (C), ESE-15-one (D) and EMBS-treated cells (E) demonstrated detyrosinated microtubule fragments. All treated cell samples demonstrated a decrease in cell density with rounded, shrunken cells. Apoptotic bodies are evident in the ESE-16 treated cells (X63 oil objective).</p
<p>LDH levels of ESE-15-one, EMBS and ESE-16 -treated HeLa cells and vehicle-treated contro... more <p>LDH levels of ESE-15-one, EMBS and ESE-16 -treated HeLa cells and vehicle-treated control cells after 24 h exposure. The background control consists of growth medium only. The low control refers to cells resuspended in growth medium and the high control to cells resuspended in growth medium with cell lysis solution added to the cells shortly before the experiment was terminated (according to the manufacturer's instructions). No statistically significant increase in LDH levels was observed in treated cells. These results showed that the sulphamoylated compounds were not toxic to the cells.</p
<p>Flow cytometry was conducted to present the effects of these sulphamoylated compounds on... more <p>Flow cytometry was conducted to present the effects of these sulphamoylated compounds on cytochrome <i>c</i> release from the mitochondria into the cytoplasm. Vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate-treated cells (B), ESE-16-treated cells (C), ESE-15-one-treated cells (D) and EMBS-treated cells (E) following 24 h of exposure. All three compound-treated cells demonstrated increased levels of cytochrome <i>c</i> release when compared to the vehicle-treated cells.</p
<p>Caspase 8 and caspase 6 activity ratios of compound- and actinomycin D-treated cells com... more <p>Caspase 8 and caspase 6 activity ratios of compound- and actinomycin D-treated cells compared to vehicle-treated cells. Caspase 6 and caspase 8 activities in all compound-treated cells increased when compared to vehicle-treated cells. EMBS-treated cells demonstrated the most prominent increase in caspase 8 activity and ESE-16-treated cells the most prominent increase in caspase 6 activity when compared to vehicle-treated cells. 2-Methoxyestradiol-bis-sulphamate-treated cells are illustrated in the figure as 2MEBM due to limited space. An asterisk (*) indicates a statistically significant <i>P</i>-value of <0.05 when compared to vehicle-treated cells.</p
<p>HeLa cell numbers expressed as a % of cells relative to the control (cells propagated in... more <p>HeLa cell numbers expressed as a % of cells relative to the control (cells propagated in growth medium) after exposure to ESE-15-one, EMBS and ESE-16 for 24 h. An asterisk (*) indicates a statistically significant <i>P</i>-value of <0.05 when compared to cells propagated in growth medium.</p
<p>TEM revealed vehicle-treated cells displaying no signs of distress (A). Cells treated wi... more <p>TEM revealed vehicle-treated cells displaying no signs of distress (A). Cells treated with 2-methoxyestradiol-bis-sulphamate (B), ESE-16 (C) and ESE-15-one (D) demonstrated the occurrence of apoptotic bodies and vacuole formation. Cells treated with EMBS (E) displayed an increase in the number of vacuoles.</p
<p>Spectrophotometry results of caspase 3 activity indicated that after exposure to these s... more <p>Spectrophotometry results of caspase 3 activity indicated that after exposure to these sulphamoylated compounds caspase 3 activity increased significantly. An asterisk (*) indicates a statistically significant <i>P</i>-value <0.05 when compared to cells propagated in growth medium.</p
<p>Tubulin was incubated with the compounds ((A), ESE-16 (B), ESE-15-one (C) and EMBS (D)),... more <p>Tubulin was incubated with the compounds ((A), ESE-16 (B), ESE-15-one (C) and EMBS (D)), as described in the methods section. This assay showed that ESE-16 had a pronounced effect comparable with that of colchicine.</p
<p>Mitotracker-stained vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate... more <p>Mitotracker-stained vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate-treated cells (B), ESE-16-treated cells (C), ESE-15-one-treated cells (D) and EMBS-treated cells (E) after 24 h exposure. An increase in the number of cells with reduced mitochondrial potential treated with 2ME2 analogues compared to the vehicle-treated control cells was observed.</p
<p>Double immunoflourecence was conducted to determine the compounds' effect after 24 h... more <p>Double immunoflourecence was conducted to determine the compounds' effect after 24 h on microtubule dynamics within HeLa cells. Tyrosinated (dynamic) microtubules are visualized as red, whereas the detyrosinated (stable or stabilized) microtubules are stained in green. The vehicle-treated cells (A) demonstrated an intact dynamic microtubule structure. Both the colchicine positive control (B), as well as the ESE-16-treated cells (C) showed complete microtubule depolymerisation with few detyrosinated microtubule fragments remaining. ESE-15-one-treated cells (D) demonstrated altered microtubule morphology. EMBS-exposed cells (E) revealed fragmented microtubules. All treated cell samples demonstrated a decrease in cell density and rounded cells (X63 oil objective).</p
<p>Mitocapture-stained vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate... more <p>Mitocapture-stained vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate-treated cells (B), ESE-16-treated cells (C), ESE-15-one-treated cells (D) and EMBS-treated cells (E) after 24 h exposure. An increase in the number of cells presenting with compromised mitochondrial potential was demonstrated in all three compound-treated cells when compared to the vehicle-treated control cells.</p
<p>Flow cytometry was used to show the effects of these compounds on cytochrome <i>c&... more <p>Flow cytometry was used to show the effects of these compounds on cytochrome <i>c</i> release from the mitochondria into the cytoplasm in HeLa cells. Vehicle-treated control cells (A), 2-methoxyestradiol-bis-sulphamate-treated cells (B), ESE-16-treated cells (C), ESE-15-one-treated cells (D) and EMBS-treated cells (E) following 24 h of exposure. Cytochrome <i>c</i> release was evident in all three compound-treated cells when compared to the vehicle-treated cells.</p
2-Methoxyestradiol (2ME2) is a natural metabolite of 17-β-estradiol exerting both antiproliferati... more 2-Methoxyestradiol (2ME2) is a natural metabolite of 17-β-estradiol exerting both antiproliferative and antiangiogenic characteristics. Due to limited bioavailability, analogues of 2ME2 were designed, synthesized and evaluated for improved in vitro antiproliferative activity as well as bioavailability. 2-Methoxyestradiol-bis-sulphamate (2-MeOE2bisMATE) is an analogue of 2ME2 which was produced by sulphamoylation of the two hydroxyl groups on carbons 3 and 17. The aim of this in vitro study was to evaluate the influence of 2MeOE2bisMATE on morphology, apoptosis and autophagy induction in an oesophageal carcinoma (SNO) cell line by means of transmission electron microscopy (TEM) and flow cytometry (cyto-ID and LC3 autophagy detection assays). In 2-MeOE2bisMATE-treated cells, features of both apoptosis and autophagy such as the presence of apoptotic bodies and autophagy vesicles were observed using the transmission electron microscopy. A significant increase in the quantity of autophag...
Current chemotherapeutic agents have many side effects and are toxic to normal cells, providing i... more Current chemotherapeutic agents have many side effects and are toxic to normal cells, providing impetus to identify agents that can effectively eliminate tumorigenic cells without damaging healthy cells. The aim of this study was to examine whether combining a novel BRD4 inhibitor, ITH-47, with the antimitotic estradiol analogue, ESE-15-ol, would have a synergistic effect on inhibiting the growth of two different breast cancer cell lines in vitro. Our docking and molecular dynamics studies showed that compared to JQ1, ITH-47 showed a similar binding mode with hydrogen bonds forming between the ligand nitrogens of the pyrazole, ASN99, and water of the BRD4 protein. Data from cell growth studies revealed that the GI50 of ITH-47 and ESE-15-ol after 48 hours of exposure was determined to be 15 μM and 70 nM, respectively, in metastatic MDA-MB-231 breast cancer cells. In tumorigenic MCF-7 breast cancer cells, the GI50 of ITH-47 and ESE-15-ol was 75 μM and 60 nM, respectively, after 48 hou...
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2012
In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel ... more In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17-beta-estradiol analog in mcf-7 breast adenocarcinoma cells. After a 24 hour exposure time, cells both apoptosis and autophagy were induced.
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2013
In vitro evaluering van 2-metoksieestradiol-bis-sulfamaat op selgroei, morfologie, selsiklus en d... more In vitro evaluering van 2-metoksieestradiol-bis-sulfamaat op selgroei, morfologie, selsiklus en die moontlike induksie van tipe seldood in 'n esofagus karsinoom senyn', Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie 32(1), Art.
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2014
2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19) induces cell death via the autophagy in b... more 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19) induces cell death via the autophagy in breast adenocarcinoma cells. C19 induces cell death via autophagy in breast adenocarcinoma cells by increasing the number of acidic vacuoles formed and an increase in ROS generation, as well as accumulation of autophagosomes.
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2012
Using a novel synthesised sulphamoylated 2-methoxyestradiol analogue, C19, two types of cell deat... more Using a novel synthesised sulphamoylated 2-methoxyestradiol analogue, C19, two types of cell death, namely apoptosis and autophagy, were demonstrated in vitro when cervical cancer HeLa cells were exposed to this compound.
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 2014
Effects of estradiol analogues on blood: A pilot study. This study demonstrated that both ESE-15-... more Effects of estradiol analogues on blood: A pilot study. This study demonstrated that both ESE-15-ol en ESE-16 have no effect on blood cells. Future ex vivo and in vivo studies into the mechanism of these potentially anticancer drugs are warranted.
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