An in vitro evaluation of anti-breast cancer activity of novel, heterocyclic aromatic compounds in combination with curcumin
- Authors: Pereira, Melanie Claire
- Date: 2017
- Subjects: Breast -- Cancer Heterocyclic compounds
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/20416 , vital:29284
- Description: Nearly two thirds of breast cancers are classified as estrogen receptor positive. Estrogen receptor (+) breast cancer is usually treated with anti-estrogen therapy, using Tamoxifen as the primary standard of treatment. Unfortunately, resistance to Tamoxifen is known to occur after 1 to 3 years of Tamoxifen therapy. For this reason, improved treatment strategies for ER (+) breast cancer are urgently needed. The general toxicity exerted by most anti-cancer drugs on proliferating cells, as well as some normal cells, restricts their therapeutic use. Novel cytotoxic agents developed with unique mechanisms of action have not been therapeutically suitable, since many of these compounds lack tumour selectivity. The adverse effects and the inefficacies of most chemotherapeutic therapies have motivated extensive investigations of alternatives. Numerous studies have demonstrated the advantages of using combination therapy with naturally-derived agents as an alternative, due to the higher therapeutic efficacy; with the added benefit of lower drug usage and reduction in drug-resistance development. An excellent example of a naturally derived and common dietary agent is curcumin, the active constituent of turmeric. Curcumin is known to modulate several signalling pathways in addition to displaying a diverse range of anti-tumour activities against a number of cancer cells. Several reports point to curcumin being beneficial if used in addition to chemotherapeutic drugs. In this study, synthetic aminonaphthoquinone derivatives (coded Rau 008, Rau 010, Rau 015 and Rau 018) were tested individually and in combination with curcumin for the potential as anti-breast cancer agents in different tumour cell lines. Notably, the aminonaphthoquinone class of compounds have shown potential as anti-cancer agents in various tumour cell lines. This study was thus aimed at screening the Rau compounds (and selected combinations with curcumin) for anti-cancer activity using a range of in vitro biological assays, and was not mechanistic in nature. The IC50 values of the individual and combined drugs were determined from dose-response curves using non-linear regression analyses. Synergistic, additive and antagonistic drug interactions were assessed using combination index and isobologram approaches. Based on these interactions, selected drug combinations were then further analysed for their potential anti-estrogenic, cytotoxic, anti-angiogenic and anti-metastatic effects against the ER(-) MDA-MB-231 and ER(+) MCF-7 breast cancer cell lines. The potential anti-cancer effects of the selected drug combinations on other estrogen-responsive models, such as the osteosarcoma (MG-63) and endometrial cancer (HEC-1A) cell lines, were also investigated. Overall, the findings of this study indicated that the sensitivity of the tumour cells to the various test compounds was significantly enhanced when combined with curcumin. For instance, in the presence of curcumin, Rau 008 or Rau 010 reduced the metastatic capability of ER(-) breast cancer cells significantly. The anti-proliferative effect of the Rau 015+curcumin combination was enhanced in ER(+) and ER(-) breast cancer, including oteosarcoma cells, while a combination of Rau 015 and curcumin induced a significant cytotoxic effect in ER(-) breast cancer and endometrial cancer-derived cells. A combination of Rau 018 and curcumin inhibited the proliferation of ER(+) breast cancer, and the combined effect was significantly more enhanced compared to individual treatment. A similar effect was noted in osteosarcoma-derived cells. Further, the Rau 018+curcumin combination exerted a marked cytotoxic and anti-angiogenic effect in ER(-) breast cancer, osteosarcoma and endometrial cancer in addition to inhibiting the attachment of ER(-) breast cancer cells to collagen. Notably, curcumin enhanced the anti-proliferative effect of Tamoxifen in breast cancer (MCF-7 and MDA-MB-231), osteosarcoma and endometrial cancer, and also induced a significant cytotoxic effect against ER(+) breast cancer and osteosarcoma, whilst also reducing the invasive potential of ER(-) breast cancer. A combination of 17β-estradiol and curcumin induced a marked anti-proliferative effect in ER(+) and ER(-) breast cancer, reduced the adhesion of ER(-) breast cancer to laminin, and elicited a significant cytotoxic and anti-angiogenic effect against endometrial cancer. Notably all the selected combinations (except Rau 018 and curcumin) inhibited bone mineralization. These findings point to the vital influence of curcumin in the responsiveness of ER(+) and ER(-) tumours towards the relative test agents. Further, among the Rau+curcumin combinations tested in this study, the anti-cancer activity of Rau 015+curcumin and Rau 018+curcumin appeared to be the most effective in the different cell lines tested. Nonetheless, it appears that a combination of 30 μM Rau 018 with 100 μM curcumin may be more promising as an anti-breast cancer agent against ER(+) and perhaps triple negative breast cancer, and warrants further investigation.
- Full Text:
- Date Issued: 2017
- Authors: Pereira, Melanie Claire
- Date: 2017
- Subjects: Breast -- Cancer Heterocyclic compounds
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/20416 , vital:29284
- Description: Nearly two thirds of breast cancers are classified as estrogen receptor positive. Estrogen receptor (+) breast cancer is usually treated with anti-estrogen therapy, using Tamoxifen as the primary standard of treatment. Unfortunately, resistance to Tamoxifen is known to occur after 1 to 3 years of Tamoxifen therapy. For this reason, improved treatment strategies for ER (+) breast cancer are urgently needed. The general toxicity exerted by most anti-cancer drugs on proliferating cells, as well as some normal cells, restricts their therapeutic use. Novel cytotoxic agents developed with unique mechanisms of action have not been therapeutically suitable, since many of these compounds lack tumour selectivity. The adverse effects and the inefficacies of most chemotherapeutic therapies have motivated extensive investigations of alternatives. Numerous studies have demonstrated the advantages of using combination therapy with naturally-derived agents as an alternative, due to the higher therapeutic efficacy; with the added benefit of lower drug usage and reduction in drug-resistance development. An excellent example of a naturally derived and common dietary agent is curcumin, the active constituent of turmeric. Curcumin is known to modulate several signalling pathways in addition to displaying a diverse range of anti-tumour activities against a number of cancer cells. Several reports point to curcumin being beneficial if used in addition to chemotherapeutic drugs. In this study, synthetic aminonaphthoquinone derivatives (coded Rau 008, Rau 010, Rau 015 and Rau 018) were tested individually and in combination with curcumin for the potential as anti-breast cancer agents in different tumour cell lines. Notably, the aminonaphthoquinone class of compounds have shown potential as anti-cancer agents in various tumour cell lines. This study was thus aimed at screening the Rau compounds (and selected combinations with curcumin) for anti-cancer activity using a range of in vitro biological assays, and was not mechanistic in nature. The IC50 values of the individual and combined drugs were determined from dose-response curves using non-linear regression analyses. Synergistic, additive and antagonistic drug interactions were assessed using combination index and isobologram approaches. Based on these interactions, selected drug combinations were then further analysed for their potential anti-estrogenic, cytotoxic, anti-angiogenic and anti-metastatic effects against the ER(-) MDA-MB-231 and ER(+) MCF-7 breast cancer cell lines. The potential anti-cancer effects of the selected drug combinations on other estrogen-responsive models, such as the osteosarcoma (MG-63) and endometrial cancer (HEC-1A) cell lines, were also investigated. Overall, the findings of this study indicated that the sensitivity of the tumour cells to the various test compounds was significantly enhanced when combined with curcumin. For instance, in the presence of curcumin, Rau 008 or Rau 010 reduced the metastatic capability of ER(-) breast cancer cells significantly. The anti-proliferative effect of the Rau 015+curcumin combination was enhanced in ER(+) and ER(-) breast cancer, including oteosarcoma cells, while a combination of Rau 015 and curcumin induced a significant cytotoxic effect in ER(-) breast cancer and endometrial cancer-derived cells. A combination of Rau 018 and curcumin inhibited the proliferation of ER(+) breast cancer, and the combined effect was significantly more enhanced compared to individual treatment. A similar effect was noted in osteosarcoma-derived cells. Further, the Rau 018+curcumin combination exerted a marked cytotoxic and anti-angiogenic effect in ER(-) breast cancer, osteosarcoma and endometrial cancer in addition to inhibiting the attachment of ER(-) breast cancer cells to collagen. Notably, curcumin enhanced the anti-proliferative effect of Tamoxifen in breast cancer (MCF-7 and MDA-MB-231), osteosarcoma and endometrial cancer, and also induced a significant cytotoxic effect against ER(+) breast cancer and osteosarcoma, whilst also reducing the invasive potential of ER(-) breast cancer. A combination of 17β-estradiol and curcumin induced a marked anti-proliferative effect in ER(+) and ER(-) breast cancer, reduced the adhesion of ER(-) breast cancer to laminin, and elicited a significant cytotoxic and anti-angiogenic effect against endometrial cancer. Notably all the selected combinations (except Rau 018 and curcumin) inhibited bone mineralization. These findings point to the vital influence of curcumin in the responsiveness of ER(+) and ER(-) tumours towards the relative test agents. Further, among the Rau+curcumin combinations tested in this study, the anti-cancer activity of Rau 015+curcumin and Rau 018+curcumin appeared to be the most effective in the different cell lines tested. Nonetheless, it appears that a combination of 30 μM Rau 018 with 100 μM curcumin may be more promising as an anti-breast cancer agent against ER(+) and perhaps triple negative breast cancer, and warrants further investigation.
- Full Text:
- Date Issued: 2017
The molecular analysis of the interation surface between sCD23 and the B2-integrins, CD11b & CD11c
- Authors: Pereira, Melanie Claire
- Date: 2012
- Subjects: CD23 antigen , Immune response -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10337 , http://hdl.handle.net/10948/d1014734
- Description: Both CD23 and the β2 integrins (also known as CD11/CD18) have very important immunological functions, especially during the allergic response where the binding of CD23 to β2 integrins contributes to various types of signalling in monocytes which can result in drastic sensitivities experienced by some allergic individuals. CD23, also known as the low affinity receptor for immunoglobulin E or (FcεRII), is a type II transmembrane glycoprotein which is synthesized by haematopoietic cells and has biological activity in both membrane-bound and freely soluble forms. It acts via a number of receptors, including the β2 integrins. β2 integrins are specifically found on leukocytes and they play important roles in cell–cell or cell–matrix adhesion via their ability to bind multiple ligands. These molecules occur as heterodimers consisting of an alpha (α) and beta (β) subunit. The α-subunits of β2 integrins contain an approximately 200-amino-acid inserted domain or I-domain which is implicated in ligand binding function. There are four different types of β2 integrins, namely CD11a, CD11b, CD11c and CD11d, all dimers with the common beta subunit, CD18. CD23 and CD11/18 are natural ligands of each other; however the interaction site for CD23 is unknown. It is postulated that the integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N-terminus of the lectin head region of CD23, which is focussed around Arg172, Lys173 and Cys174 (RKC). This study thus focused on the interaction between the I-domain of CD11 (b and c) and a recombinant 25kDa construct of sCD23. In order to understand the characteristics of ligand binding between the relevant proteins of interest, alanine substitutions on the RKC motif of CD23 were made via site-directed mutagenesis. Consequently, a recombinant form of the I-domain of CD11 (b and c) as well as a wild type (containing the RKC motif) and mutant form (containing an AAC motif) of sCD23 were expressed and purified. The CD11 recombinant proteins were purified via affinity chromatography and the CD23 recombinant proteins via gel filtration chromatography. In addition, synthetic (CD23 derived) peptides, one containing the RKC sequence and the other the AAC sequence, were designed and custom synthesized. The synthetic peptides as well as the recombinant CD23 proteins were then analyzed for their interaction with the CD11 I-domain via ELISA. Subsequent ELISA analyses showed that the native sCD23 and the RKC peptide were able to bind to the integrin α I-domain whereas the mutant sCD23 and the corresponding synthetic AAC peptide failed to bind. This interaction was also analysed via flow cytometry using differentiated U937 cells, yielding similar results. ELISA analyses for the sCD23-CD11b I-domain interaction showed a Kd of 0.36 ± 0.14 μM whereas the RKC-CD11b I-domain interaction yielded a Kd of 1.75 ± 0.58 μM. Similarly, the sCD23-CD11c I-domain interaction yielded a Kd of 0.39 ± 0.09 μM and 1.53 ± 0.72 μM for the RKC-CD11c I-domain interaction. Peptide inhibitory analysis, analysed via ELISA and flow cytometry, reinforced the fact that the RKC motif on sCD23 is a prerequisite for ligand binding of the CD11b/c I-domain.
- Full Text:
- Date Issued: 2012
- Authors: Pereira, Melanie Claire
- Date: 2012
- Subjects: CD23 antigen , Immune response -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10337 , http://hdl.handle.net/10948/d1014734
- Description: Both CD23 and the β2 integrins (also known as CD11/CD18) have very important immunological functions, especially during the allergic response where the binding of CD23 to β2 integrins contributes to various types of signalling in monocytes which can result in drastic sensitivities experienced by some allergic individuals. CD23, also known as the low affinity receptor for immunoglobulin E or (FcεRII), is a type II transmembrane glycoprotein which is synthesized by haematopoietic cells and has biological activity in both membrane-bound and freely soluble forms. It acts via a number of receptors, including the β2 integrins. β2 integrins are specifically found on leukocytes and they play important roles in cell–cell or cell–matrix adhesion via their ability to bind multiple ligands. These molecules occur as heterodimers consisting of an alpha (α) and beta (β) subunit. The α-subunits of β2 integrins contain an approximately 200-amino-acid inserted domain or I-domain which is implicated in ligand binding function. There are four different types of β2 integrins, namely CD11a, CD11b, CD11c and CD11d, all dimers with the common beta subunit, CD18. CD23 and CD11/18 are natural ligands of each other; however the interaction site for CD23 is unknown. It is postulated that the integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N-terminus of the lectin head region of CD23, which is focussed around Arg172, Lys173 and Cys174 (RKC). This study thus focused on the interaction between the I-domain of CD11 (b and c) and a recombinant 25kDa construct of sCD23. In order to understand the characteristics of ligand binding between the relevant proteins of interest, alanine substitutions on the RKC motif of CD23 were made via site-directed mutagenesis. Consequently, a recombinant form of the I-domain of CD11 (b and c) as well as a wild type (containing the RKC motif) and mutant form (containing an AAC motif) of sCD23 were expressed and purified. The CD11 recombinant proteins were purified via affinity chromatography and the CD23 recombinant proteins via gel filtration chromatography. In addition, synthetic (CD23 derived) peptides, one containing the RKC sequence and the other the AAC sequence, were designed and custom synthesized. The synthetic peptides as well as the recombinant CD23 proteins were then analyzed for their interaction with the CD11 I-domain via ELISA. Subsequent ELISA analyses showed that the native sCD23 and the RKC peptide were able to bind to the integrin α I-domain whereas the mutant sCD23 and the corresponding synthetic AAC peptide failed to bind. This interaction was also analysed via flow cytometry using differentiated U937 cells, yielding similar results. ELISA analyses for the sCD23-CD11b I-domain interaction showed a Kd of 0.36 ± 0.14 μM whereas the RKC-CD11b I-domain interaction yielded a Kd of 1.75 ± 0.58 μM. Similarly, the sCD23-CD11c I-domain interaction yielded a Kd of 0.39 ± 0.09 μM and 1.53 ± 0.72 μM for the RKC-CD11c I-domain interaction. Peptide inhibitory analysis, analysed via ELISA and flow cytometry, reinforced the fact that the RKC motif on sCD23 is a prerequisite for ligand binding of the CD11b/c I-domain.
- Full Text:
- Date Issued: 2012
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