Lesetja Raymond Motadi

Silencing RBBP6 (Retinoblastoma Binding Protein 6) sensitises breast cancer cells MCF7 to staurosporine and camptothecin-induced cell death

Retinoblastoma Binding Protein 6 (RBBP6) is a multi-domain protein that uses its ring finger domain to interact with p53 and pRb tumour suppressor genes. The mechanism by which RBBP6 uses to degrade p53 is still unknown; nonetheless it is well known that RBBP6 promotes cell proliferation in several cancers by negatively regulating p53 via its E3 ubiquitin ligase activity. Degradation of p53 by RBBP6 may compromise p53-mediated apoptosis in breast cancer. This study is intended to investigate, the potential applications of RNA interference (RNAi) to block RBBP6 expression, as well as its subsequent effect on cell growth and apoptosis. Our studies indicate that the knockdown of RBBP6 by siRNA modulates p53 gene expression involved in cell death pathways and apoptosis, showing statistically significant gene expression differences. RBBP6 siRNA significantly reduced cell growth compared to the control samples and inhibition of cellular proliferation was observed between 24 and 48h, as shown in the data obtained by real time cell analysis using the xCELLigence system. These results were further confirmed by flow cytometer which showed some apoptotic activity. About 20.7% increase in apoptosis was observed in cells co-treated with RBBP6 siRNA and camptothecin when compared to camptothecin-only whereas in siRBBP6 and staurosporine treated cells there was only an 8.8% increase in apoptosis. These findings suggest that silencing RBBP6 may be a novel strategy to promote camptothecin-induced apoptosis in breast cancer cells.

RBBP6: a potential biomarker of apoptosis induction in human cervical cancer cell lines

Overexpression of RBBP6 in cancers of the colon, lung, and esophagus makes it a potential target in anticancer therapy. This is especially important because RBBP6 associates with the tumor suppressor gene p53, the inactivation of which has been linked to over 50% of all cancer types. However, the expression of RBBP6 in cancer and its interaction with p53 are yet to be understood in order to determine whether or not RBBP6 is cancer promoting and therefore a potential biomarker. In this study, we manipulated RBBP6 expression levels followed by treatment with either camptothecin or γ-aminobutyric acid in cervical cancer cells to induce apoptosis or cell cycle arrest. We began by staining human cervical cancer tissue sections with anti-RBBP6 monoclonal antibody to evaluate the extent of expression of RBBP6 in patients’ specimens. We followed on with silencing the overexpression of RBBP6 and treatment with anticancer agents to evaluate how the specimens respond to combinational therapy. Apoptosis induction was evaluated through confocal microscope, and flow cytometry using annexin V staining, and also by checking the mitochondrial and caspase-3/7 activity. Cell cycle arrest was evaluated using flow cytometry through staining with propidium iodide. RBBP6 was highly expressed in cervical cancer tissue sections that were in stage II or III of development. Silencing RBBP6 followed by treatment with γ-aminobutyric acid and camptothecin seems to sensitize cells to apoptosis induction rather than cell cycle arrest. Overexpression of RBBP6 seems to promote S-phase in cell cycle and cell proliferation. These results predict a proliferative role of RBBP6 in cancer progression rather than as a cancer-causing gene. Furthermore, sensitization of cells to camptothecin-induced apoptosis by RBBP6 targeting suggests a promising tool for halting cervical cancer progression.

RBBP6 expressional effects on cell proliferation and apoptosis in breast cancer cell lines with distinct p53 statuses

Introduction Breast cancer is the most common malignancy amongst women and has a higher incidence rate than lung cancer. Its tumor progression partially results from inactivation of p53 which is caused by overexpression of ubiquitous regulatory proteins possessing p53-binding domain. RBBP6 is regarded as one of the ubiquitous proteins because of its RING finger-like domain which enables it to possess E3 ligase activity. Thus, it has become a potential target in cancer treatment as it is highly expressed in various malignancies including cancer. However, it is not clearly defined whether the effect of RBBP6 on cell growth and apoptosis is cell line-dependent, more especially in breast cancer cell lines that have distinct p53 expression profiles. This study aims at evaluating the effects of RBBP6 on cell growth and apoptosis in breast cancer cell lines with different p53 expressions. Methods Following the analysis at mRNA and protein levels in breast cancer tissue, RBBP6 expression was successfully manipulated using gene silencing and protein overexpression techniques in MCF-7 and MDA-MB-231 cell lines. The cells were co-treated with siRBBP6 and anticancer agents following apoptosis detection, which was confirmed by caspase 3/7 activity and quantification of apoptotic genes. Results RBBP6 was overexpressed in breast cancer tissues that were classified as stages 3 and 4, while in stage 1, its expression was much lower. The MCF-7 cell line which expresses wild-type p53 was more sensitive to apoptosis induction than MDA-MB-231 which is a mutant p53-expressing cell line. These data suggest that RBBP6 silencing triggers significant levels of intrinsic apoptosis, and its overexpression appears to promote cell proliferation in wild-type p53-expressing MCF-7 cell line as opposed to MDA-MB-231 cells. Conclusion The effect of RBBP6 on cell proliferation and apoptosis induction in breast cancer seems to be cell line-dependent based on p53 status.

Abstract 3517: Expression studies of RBBP6 in breast and cervical cancer suggests a role in apoptosis activation

Ubiquitin-like DWNN domain and RING finger-like domain presence on Retinoblastoma Binding protein 6 (RBBP6), is linked to its function as cancer promoting gene through p53 degradation via its p53-binding domain. RBBP6 bind and regulate the expression of p53 and pRB in the cell machinery implicates RBBP6 in cell proliferation during cancer development (Moela et al 2014). Studies indicate that overexpression of the mouse spliced variants led to apoptosis and cell cycle arrest whilst deregulation in mice led to embryonic lethality followed by p53 accumulation and a widespread apoptosis (Li et al., 2007). In this study, focuses on determining the expression pattern of RBBP6 in human tissue models of breast and cervical cancer. The study further aims to analyze apoptosis and cell cycle following either knockdown or overexpression of RBBP6 coupled to treatment with potential anticancer compounds, camptothecin and GABA. Quantitative analysis by qPCR in HeLa, SiHa, MCF-7and MDA-MB-231 cell lines shows that RBBP6 expression is fairly high in cancer cells when compared to normal lung fibroblast. Following co-treatment with GABA and cDNA or siRNA, in cDNA treated there was a reduction in both apoptosis as measured by flow cytometer and TP53 expression while in siRNA there was increased expression of TP53 and those of Bax and Bak1 which seemed to account for increased apoptosis. Cell cycle presented increase in G0/G1 and the G2/M following treatment with siRNA and GABA. GABA alone could not induce a significant increase in apoptotic cells whereas when combined with siRNA it was able to induce over 30% apoptosis. These results suggest that RBBP6 might be a cancer promoting gene. Citation Format: Mpho Choene, Pontsho Moela, Lesetja Motadi. Expression studies of RBBP6 in breast and cervical cancer suggests a role in apoptosis activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3517.

Abstract 2190: Isolated cannabidiol from Cannabis sativa plant extracts inhibit growth and induce apoptosis in cervical cancer cells

Cervical cancer remains a global health related issue among females of Sub-Saharan Africa, with over half a million new cases reported each year. Different therapeutic regimens have been suggested in various regions of Africa, however, over a quarter of a million women die of cervical cancer, annually. Therefore, it is important to search for new drugs through effective screening of medicinal plant extracts to identify lead anti-cervical cancer drugs. The aim of this study was to evaluate for the anti-growth effects of Cannabis sativa extracts and its isolate, cannabidiol on cervical cancer cell lines HeLa, SiHa, and ME-180. To determine for the presence of important constituents and evaluate for the anti-growth effects, phytochemical screening, MTT assay, cell growth analysis, flow cytometry, morphology analysis, Western blot, caspase 3/7 assay, and ATP measurement assay were conducted were conducted. Results obtained indicate that both plant extracts induced cell death at an IC50 of 50 - 100μg/ml and the Inhibition of cell growth was cell line dependent. Flow cytometry confirmed that, with or without cell cycle arrest, the type of induced cell death was apoptosis. Cannabis sativa extracts led to the up-regulation of apoptosis proteins (p53, Bax, caspase-3, and caspase-9) and the down regulation of anti-apoptosis proteins (Bcl-2 and RBBP6), signalling the execution of apoptosis. Apoptosis induction was further confirmed by morphological changes, an increase in Caspase 3/7 and a decrease in the ATP levels. In conclusion, this data implies Cannabis sativa crude extracts has the potential to inhibit growth and induce apoptosis in cervical cancer cell lines, which may be due to the presence of cannabidiol. Citation Format: Lesetja Raymond Motadi, sindiswa lukhelo. Isolated cannabidiol from Cannabis sativa plant extracts inhibit growth and induce apoptosis in cervical cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2190.

Apoptotic Molecular Advances in Breast Cancer Management

Breast cancer is the most common cancer type amongst women, accounting for most fe‐ male cancer deaths second to cervical cancer worldwide. It is, therefore, highly crucial to understand the molecular biology and explore other pathways involved in carcinogenesis in order to select appropriate treatment not only for breast cancer but for other cancers as well. Cancer progression is favoured by DNA damage and in most cases a consequent disruption of the apoptotic pathway, thus leading to uncontrolled cell proliferation. Therefore, current therapeutic strategies aim at targeting the apoptotic pathways in order to combat cancer. In this manuscript, we discuss the ways in which evasion of apoptosis during carcinogenesis occurs and the types of current therapeutic strategies as well as promising future approaches against breast cancer.

Abstract 5543: Elucidating the cytotoxic effects of taxol,camptothecin and Lobostemon fruticosus extracts on non-small cell lung cancer

Lung cancer is reportedly the most common form of cancer worldwide, with a 13-15% occurrence of all known cancers being reported daily. Lung cancer is also the most deadly form of cancer as it has been observed to be resistant to most therapeutic treatments and it demonstrates a metastasis within lymph nodes. An alternative approach is to identify the cytotoxic effects of non-invasive medicinal plant extracts on NSCLC. Thus the aim of the study was to compare the cytotoxic effects of the invasive anti-cancer drugs, taxol and camptothecin, with less invasive plant extracts of L. fruticosus on cell line A549. Cells seem to show a degree of cell death after treatment however some degree of cells still remain viable indicating usefulness in chemotherapy. The following techniques were used, exCELLigence RTCA, Cell cycle, Annexin V apoptosis analysis and LC-MS to identify the compound. Cells treated with camptothecin, Methanol and butanol extracts indicate some degree of early apoptosis which was indicative of little to no DNA fragmentation. Cell cycle arrest was observed in both camptothecin and taxol treated cells, however there was little evidence to suggest the same for L. Fruticosus extracts as there was a high degree of cell death that was observed during the cell cycle analysis. Apoptotic genes that were found in A549 treated cells upregulated in the absence of p53. Phytochemical screening of the L. Fruticosus plant extracts indicated that the plant possesses compounds that display apoptotic activity. These findings suggest that camptothecin, taxol and extract of L. fruticosus display cytotoxic activity on A549 cells. The upregulation of genes in absence of p53 seem to suggest that treatments induce apoptosis using an alternative apoptotic pathway. Note: This abstract was not presented at the meeting. Citation Format: Lesetja Raymond Motadi, Lungile Ndlovu. Elucidating the cytotoxic effects of taxol,camptothecin and Lobostemon fruticosus extracts on non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5543. doi:10.1158/1538-7445.AM2015-5543

Abstract 1786: Role of Euphorbia tirucalli terpenes cocktail extracts in the regulation of molecular cell death: implications in gynecological cancer progression

Gynaecological cancers are increasingly becoming the cause of death in females in developing countries. This type of cancers, especially cervical cancer is in the increase due to co-infection with HIV. Many of these people are solely dependent on traditional remedies, as conventional treatment might be expensive or inaccessible to them. In this study we characterised butanolic, hexane and methanolic extracts of Euphorbia tirucalli using both TLC and LC-MS spectroscopy. Following extract analysis, we looked at the effects of the extracts in regulating cell death in breast (MDA-MB 231), ovarian (RMG-1) and cervical (SiHa) cancer cell lines. The cells were treated with varying concentrations of the cocktailed extracts for up to 48 hours. A real time cell analyzer (xCELLigence) was used to monitor cell growth and to determine IC50. MTT assay was used to assess cell viability 24 hours post treatment with extracts. To determine if the extracts had an effect on cell cycle arrest, cell cycle distribution was determined by staining treated cancer cells with propidium iodide and the percentages of G0/G1, S, G2/M cell populations were analysed using flow cytometry. To determine the type of cell death induced, flow cytometry using annexin V was employed. At IC50, molecular analysis was performed to evaluate the effect cocktailed extracts had on apoptotic and cell cycle genes. Cocktailed extracts were found to inhibit cell proliferation in a concentration and cell type dependent manner. Cells were also found to have been arrested at G0/G1 and in some cases apoptosis induced. In general most pro-apoptotic genes like Bax and caspase-8 were upregulated this included the cell cycle gene p21 which was found to be significantly up-regulated in cells treated with extracts. These results suggest that the 3 cocktailed extracts might induce apoptosis or arrest cells at G0/G1 with the molecular mechanism that suggest activation of caspase-8 and Bax. Note: This abstract was not presented at the meeting. Citation Format: Mpho Choene, Lesetja Motadi. Role of Euphorbia tirucalli terpenes cocktail extracts in the regulation of molecular cell death: implications in gynecological cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1786. doi:10.1158/1538-7445.AM2015-1786

Abstract 344: Silencing RBBP6 (retinoblastoma binding protein 6) sensitizes breast cancer cells to staurosporine and camptothecin-induced cell death

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Retinoblastoma Binding Protein 6 (RBBP6) is a multi-domain protein that uses its ring finger domain to interact with p53 and pRb tumour suppressor genes. The mechanism by which RBBP6 uses to degrade p53 is still unknown; nonetheless it is well known that RBBP6 promotes cell proliferation in several cancers by negatively regulating p53 via its E3 ubiquitin ligase activity (Ntwasa, 2008). Degradation of p53 by RBBP6 may compromise p53-mediated apoptosis in breast cancer. This study is intended to investigate, the potential applications of RNA interference (RNAi) to block RBBP6 expression, as well as its subsequent effect on cell growth and apoptosis. To achieve these methodologies the following techniques were used: RT-PCR, western blotting, xCELLigence system and flow cytometry. Our studies indicate that the knockdown of RBBP6 expression by siRNA modulates p53 gene involved in cell death pathways and apoptosis, showing statistically significant gene expression differences. RBBP6 siRNA significantly reduced cell index (CI) compared to the control samples and we observed an inhibition of cellular proliferation in the interval of between 24 and 48 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. These results were further confirmed by flow cytometer which showed some apoptotic activity. About 20.7% increase in apoptosis was observed in cells co-treated with RBBP6 siRNA and camptothecin when compared to camptothecin-only whereas in siRBBP6 and Staurosporine treated there was only 8.8% increase in apoptosis. These findings suggest that silencing RBBP6 may be a novel strategy to promote staurosporine- and camptothecin-induced apoptosis in breast cancer cells. Citation Format: Lesetja Raymond Motadi, Pontsho Moela, Mpho S. Choene. Silencing RBBP6 (retinoblastoma binding protein 6) sensitizes breast cancer cells to staurosporine and camptothecin-induced cell death. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 344. doi:10.1158/1538-7445.AM2014-344

Abstract 3216: Anti-proliferative properties of Euphorbia mauritanica medicinal plant against breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Over the decades, the increased interest in the use of natural resources in pharmaceutical and phytotherapeutic research has led to manufacture of drugs (including anti-cancer drugs) derived from natural sources With our increased understanding of the molecular mechanisms underlying cancer progression and cell death, it is therefore necessary to test if there are any natural products that can be used to act as antitumor agents. As breast cancer continues to be the leading cause of cancer in females, we aimed to exploit the use of South African traditional medicine, Euphorbia mauritanica as a tool for anti breast cancer therapy. Euphorbia mauritanica was tested for anti-proliferative properties against MCF-7 and Cama-1 breast cancer cell lines. The following solvents were used to extract active compounds from E. mauritanica: n-hexane, dichloromethane, chloroform, ethyl acetate, acetone, methanol and butanol. A real time cell analyzer (Xcelligence) was used to monitor cell growth over a period of 48 hours following treatment of cells with the extracts. Butanolic extracts showed the highest level of cytotoxicity on both MCF-7 and Cama-1 cell lines with IC50 of 15 and 30 µg/ml respectively. These were followed by hexanolic extracts with IC50 of 30 and 50 µg/ml and methanolic extracts with IC50 of 50 and 100 µg/ml. Flow cytometry using annexin V was further employed to determine if cell death was as a result of apoptosis and the data confirmed that this was most likely the case. On investigating whether the observed apoptosis was via p53 mediated pathway, western blot analysis of p53, bcl-2, caspase 9, caspase3, RBBP6 and mdm2 showed that treatment with the 3 extracts did not have a significant effect on any of these genes, suggesting that their mode of apoptosis induction maybe through an alternative apoptotic pathway. Citation Format: Mpho S. Choene, Lesetja R. Motadi. Anti-proliferative properties of Euphorbia mauritanica medicinal plant against breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3216. doi:10.1158/1538-7445.AM2014-3216