Molecular Farming of Pembrolizumab and Nivolumab.

Immune checkpoint inhibitors (ICIs) are a class of immunotherapy agents capable of alleviating the immunosuppressive effects exerted by tumorigenic cells. The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is one of the most ubiquitous checkpoints utilized by tumorigenic cells for immune evasion by inducing apoptosis and inhibiting the proliferation and cytokine production of T lymphocytes. Currently, the most frequently used ICIs targeting the PD-1/PD-L1 checkpoint include monoclonal antibodies (mAbs) pembrolizumab and nivolumab that bind to PD-1 on T lymphocytes and inhibit interaction with PD-L1 on tumorigenic cells. However, pembrolizumab and nivolumab are costly, and thus their accessibility is limited in low- and middle-income countries (LMICs). Therefore, it is essential to develop novel biomanufacturing platforms capable of reducing the cost of these two therapies. Molecular farming is one such platform utilizing plants for mAb production, and it has been demonstrated to be a rapid, low-cost, and scalable platform that can be potentially implemented in LMICs to diminish the exorbitant prices, ultimately leading to a significant reduction in cancer-related mortalities within these countries.

The Biological Relevance of Papaverine in Cancer Cells.

Papaverine (PPV), a benzylisoquinoline alkaloid, extracted from the Papaverine somniferum plant, is currently in clinical use as a vasodilator. Research has shown that PPV inhibits phosphodiesterase 10A (PDE10A,) resulting in the accumulation of cyclic adenosine 3', 5'-monophosphate (cAMP) that affects multiple downstream pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), a mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF). The accumulation of cAMP can further affect mitochondrial metabolism through the activation of protein kinase A (PKA), which activates the mitochondrial complex I. Literature has shown that PPV exerts anti-proliferative affects in several tumorigenic cell lines including adenocarcinoma alveolar cancer (A549) and human hepatoma (HepG-2) cell lines. Cell cycle investigations have shown varying results with the effects dependent on concentration and cell type with data suggesting an increase in cells occupying the sub-G1 phase, which is indicative of cell death. These results suggest that PPV may be a beneficial compound to explore for the use in anticancer studies. More insight into the effects of the compound on cellular and molecular mechanisms is needed. Understanding the effects PPV may exert on tumorigenic cells may better researchers' understanding of phytomedicines and the effects of PPV and PPV-derived compounds in cancer.

In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines.

Papaverine (PPV) is a benzylisoquinoline alkaloid isolated from Papaver somniferum that exerts antiproliferative activity. However, several questions remain regarding the biochemical pathways affected by PPV in tumourigenic cells. In this study, the influence of PPV on cell migration (light microscopy), expression of vascular endothelial growth factor (VEGF) B, VEGF R1, VEGF R2, and phosphorylated focal adhesion kinase (pFAK) were investigated using spectrophotometry in MDA-MB-231-, A549- and DU145 cell lines. The migration assay revealed that, after 48 h, PPV (100 µM) reduced cell migration to 81%, 91%, and 71% in MDA-MB-231-, A549-, and DU145 cells, respectively. VEGF B expression was reduced to 0.79-, 0.71-, and 0.73-fold after 48 h of exposure to PPV in MDA-MB-231-, A549- and DU145 cells, while PPV exposure of 48 h increased VEGF R1 expression in MDA-MB-231- and DU145 cells to 1.38 and 1.46. A fold decrease in VEGF R1 expression was observed in A549 cells to 0.90 after exposure to 150 µM. No statistically significant effects were observed on VEGF R2- and FAK expression after exposure to PPV. This study contributes to the understanding of the effects of a phytomedicinal alkaloid compound in cancer cells and may provide novel approaches to the application of non-addictive alkaloids.

In Vitro Effects of Papaverine on Cell Proliferation, Reactive Oxygen Species, and Cell Cycle Progression in Cancer Cells.

Papaverine (PPV) is an alkaloid isolated from the Papaver somniferum. Research has shown that PPV inhibits proliferation. However, several questions remain regarding the effects of PPV in tumorigenic cells. In this study, the influence of PPV was investigated on the proliferation (spectrophotometry), morphology (light microscopy), oxidative stress (fluorescent microscopy), and cell cycle progression (flow cytometry) in MDA-MB-231, A549, and DU145 cell lines. Exposure to 150 µM PPV resulted in time- and dose-dependent antiproliferative activity with reduced cell growth to 56%, 53%, and 64% in the MDA-MB-231, A549, and DU145 cell lines, respectively. Light microscopy revealed that PPV exposure increased cellular protrusions in MDA-MB-231 and A549 cells to 34% and 23%. Hydrogen peroxide production increased to 1.04-, 1.02-, and 1.44-fold in PPV-treated MDA-MB-231, A549, and DU145 cells, respectively, compared to cells propagated in growth medium. Furthermore, exposure to PPV resulted in an increase of cells in the sub-G1 phase by 46% and endoreduplication by 10% compared to cells propagated in growth medium that presented with 2.8% cells in the sub-G1 phase and less than 1% in endoreduplication. The results of this study contribute to understanding of effects of PPV on cancer cell lines.

Dysregulation of Catalase by a Sulphamoylated Estradiol Analogue Culminates in Antimitotic Activity and Cell Death Induction in Breast Cancer Cell Lines.

Recent findings revealed that 2-ethyl-17-oxoestra-1,3,5(10)-trien-3-yl sulfamate (ESE-one) induces antiproliferative activity and cell rounding dependent on the generation of superoxide anion, hydrogen peroxide and peroxyl radical. In the current study, the role of these reactive oxygen species was assessed in the activity exerted by ESE-one on cell cycle progression, mitochondrial membrane potential and cell death induction in breast tumorigenic cells. The influence of ESE-one was also investigated on superoxide dismutase and catalase activity. ESE-one induced a time-dependent accumulation of cells in the G1 phase and G2/M phase that is partially impaired by tiron and trolox and N,N'-dimethylthiourea suggesting that superoxide anion, hydrogen peroxide and peroxyl radical are required for these effects exerted by ESE-one. Flow cytometry data in MCF-7 cells demonstrated that tiron decreased depolarization of the membrane potential in ESE-one exposed cells, indicating that superoxide anion plays a role in the depolarization effects induced by ESE-one. Spectrophotometry data showed that ESE-one decreased catalase activity in both cell lines. This study contributes towards pertinent information regarding the effects of an in silico-designed sulfamoylated compound on antioxidant enzymes leading to aberrant quantities of specific reactive oxygen species resulting in antimitotic activity culminating in the induction of cell death in breast cancer cell lines.

Sulphamoylated Estradiol Analogue Induces Reactive Oxygen Species Generation to Exert Its Antiproliferative Activity in Breast Cancer Cell Lines.

2-Methoxyestradiol (2ME), a 17ß-estradiol metabolite, exerts anticancer properties in vitro and in vivo. To address 2ME's low bioavailability, research led to the in silico design of sulphamoylated 2ME analogues. However, the role of oxidative stress induced in the activity exerted by sulphamoylated compounds remains elusive. In the current study, the influence of 2-Ethyl-17-oxoestra-1,3,5(10)-trien-3-yl sulphamate (ESE-one) on reactive oxygen species (ROS) induction and its effect on cell proliferation, as well as morphology, were assessed in breast tumorigenic cells (MCF-7 and MDA-MB-231). Fluorescent microscopy showed that sulphamoylated estradiol analogues induced hydrogen peroxide and superoxide anion, correlating with decreased cell growth demonstrated by spectrophotometry data. ESE-one exposure resulted in antiproliferation which was repressed by tiron (superoxide inhibitor), trolox (peroxyl inhibitor) and N,N'-dimethylthiourea (DMTU) (hydrogen peroxide inhibitor). Morphological studies demonstrated that tiron, trolox and DMTU significantly decreased the number of rounded cells and shrunken cells in MCF-7 and MDA-MB-231 cells induced by ESE-one. This in vitro study suggests that ESE-one induces growth inhibition and cell rounding by production of superoxide anion, peroxyl radical and hydrogen peroxide. Identification of these biological changes in cancer cells caused by sulphamoylated compounds hugely contributes towards improvement of anticancer strategies and the ROS-dependent cell death pathways in tumorigenic breast cells.

In vitro quantification: Long-term effect of glucose deprivation on various cancer cell lines.

OBJECTIVE: Although metabolic treatment of highly glycolytic cancers and metastases is becoming an important research field, the effects of such treatments are not fully quantified yet. In this article we attempt to quantify the effect of long-term glucose deprivation (similar to ketogenic diets) on cancer cells using in vitro tests. METHODS: Two tumorigenic cell lines were used, namely a metastatic breast and a cervical cancer cell line. The non-tumorigenic control cell line was an immortalized breast cell line. All the cell lines were stabilized at a typical average human blood glucose level of 6 mmol/L. The cell lines were then exposed to the therapeutic blood glucose level of 3 mmol/L for 90 d. RESULTS: The tests indicated that glucose deprivation restricted the different cancer cell lines' growth more than that of non-tumorigenic cells. The different cell lines were also differentially affected, which suggests that long-term glucose deprivation will not be equally effective for different types of cancer. The highly glycolytic breast cancer cell line was most adversely affected, with cell growth decreasing to 30% after 26 d. Cell growth was stable at this level for up to 22 d. Furthermore, all of the other cancer cell lines were similarly affected. CONCLUSIONS: This in vitro data could help to direct future human in vivo tests to find the most therapeutic time (cancer cells at their most vulnerable) for additional short-term adjuvant therapies. Partial recovery of proliferation occurred after 90 d. Therefore, as expected, the results also indicated that without an adjuvant treatment, full extinction cannot be reached with the proposed long-term metabolic treatment. The need for more clinical data on long-term glucose deprivation treatments for cancer is well described in the literature. This paper attempts to add to the available pool of knowledge.

Warburg effect and its role in tumourigenesis.

Glucose is a crucial molecule in energy production and produces different end products in non-tumourigenic- and tumourigenic tissue metabolism. Tumourigenic cells oxidise glucose by fermentation and generate lactate and adenosine triphosphate even in the presence of oxygen (Warburg effect). The Na+/H+-antiporter is upregulated in tumourigenic cells resulting in release of lactate- and H+ ions into the extracellular space. Accumulation of lactate- and proton ions in the extracellular space results in an acidic environment that promotes invasion and metastasis. Otto Warburg reported that tumourigenic cells have defective mitochondria that produce less energy. However, decades later it became evident that these mitochondria have adapted with alterations in mitochondrial content, structure, function and activity. Mitochondrial biogenesis and mitophagy regulate the formation of new mitochondria and degradation of defective mitochondria in order to combat accumulation of mutagenic mitochondrial deoxyribonucleic acid. Tumourigenic cells also produce increase reactive oxygen species (ROS) resulting from upregulated glycolysis leading to pathogenesis including cancer. Moderate ROS levels exert proliferative- and prosurvival signaling, while high ROS quantities induce cell death. Understanding the crosstalk between aberrant metabolism, redox regulation, mitochondrial adaptions and pH regulation provides scientific- and medical communities with new opportunities to explore cancer therapies.

Comparison of structures and cytotoxicity of mupirocin and batumin against melanoma and several other cancer cell lines.

Aim: To determine the computer-predicted anticancer activity of mupirocin and to compare its activities with those determined for another polyene antibiotic, batumin. Materials & methods: Molecular docking, cytotoxicity assays, cell microscopy and cell cycle progression were studied in cancer and nontumorigenic cell lines. Results & conclusion: Cytotoxicity of mupirocin against several cancerous cell lines was detected with the highest one (IC50 = 5.4 µg/ml) against melanoma cell line. The profile of cytotoxicity of mupirocin was similar to that reported for batumin. Nevertheless, the morphology of cells treated with these antibiotics and alterations in cell cycle progression suggested possible dissimilarity in their mechanisms of action. Selective cytotoxicity of mupirocin against melanoma cells potentiates further studies to discover nontoxic drugs for melanoma prevention.

Effects of glutamine deprivation on oxidative stress and cell survival in breast cell lines.

BACKGROUND: Tumourigenic cells modify metabolic pathways in order to facilitate increased proliferation and cell survival resulting in glucose- and glutamine addiction. Previous research indicated that glutamine deprivation resulted in potential differential activity targeting tumourigenic cells more prominently. This is ascribed to tumourigenic cells utilising increased glutamine quantities for enhanced glycolysis- and glutaminolysis. In this study, the effects exerted by glutamine deprivation on reactive oxygen species (ROS) production, mitochondrial membrane potential, cell proliferation and cell death in breast tumourigenic cell lines (MCF-7, MDA-MB-231, BT-20) and a non-tumourigenic breast cell line (MCF-10A) were investigated. RESULTS: Spectrophotometry demonstrated that glutamine deprivation resulted in decreased cell growth in a time-dependent manner. MCF-7 cell growth was decreased to 61% after 96 h of glutamine deprivation; MDA-MB-231 cell growth was decreased to 78% cell growth after 96 h of glutamine deprivation, MCF-10A cell growth was decreased 89% after 96 h of glutamine deprivation and BT-20 cell growth decreased to 86% after 24 h of glutamine deprivation and remained unchanged until 96 h of glutamine deprivation. Glutamine deprivation resulted in oxidative stress where superoxide levels were significantly elevated after 96 h in the MCF-7- and MDA-MB-231 cell lines. Time-dependent production of hydrogen peroxide was accompanied by aberrant mitochondrial membrane potential. The effects of ROS and mitochondrial membrane potential were more prominently observed in the MCF-7 cell line when compared to the MDA-MB-231-, MCF-10A- and BT-20 cell lines. Cell cycle progression revealed that glutamine deprivation resulted in a significant increase in the S-phase after 72 h of glutamine deprivation in the MCF-7 cell line. Apoptosis induction resulted in a decrease in viable cells in all cell lines following glutamine deprivation. In the MCF-7 cells, 87.61% of viable cells were present after 24 h of glutamine deprivation. CONCLUSION: This study demonstrates that glutamine deprivation resulted in decreased cell proliferation, time-dependent- and cell line-dependent ROS generation, aberrant mitochondrial membrane potential and disrupted cell cycle progression. In addition, the estrogen receptor positive MCF-7 cell line was more prominently affected. This study contributes to knowledge regarding the sensitivity of breast cancer cells and non-tumorigenic cells to glutamine deprivation.

Effects of glutamine deprivation on oxidative stress and cell survival in breast cell lines

BACKGROUND: Tumourigenic cells modify metabolic pathways In order to facilitate increased proliferation and cell survival resulting in glucose-and glutamine addiction. Previous research indicated that glutamine deprivation resulted in potential differential activity targeting tumourigenic cells more prominently. This is ascribed to tumourigenic cells utilising increased glutamine quantities for enhanced glycolysis-and glutaminolysis. In this study, the effects exerted by glutamine deprivation on reactive oxygen species (ROS) production, mitochondrial membrane potential, cell proliferation and cell death in breast tumourigenic cell lines (MCF-7, MDA-MB-231, BT-20) and a non-tumourigenic breast cell line (MCF-10A) were investigated. RESULTS: Spectrophotometry demonstrated that glutamine deprivation resulted in decreased cell growth in a time-dependent manner. MCF-7 cell growth was decreased to 61% after 96 h of glutamine deprivation; MDA-MB-231 cell growth was decreased to 78% cell growth after 96 h of glutamine deprivation, MCF-10A cell growth was decreased 89% after 96 h of glutamine deprivation and BT-20 cell growth decreased to 86% after 24 h of glutamine deprivation and remained unchanged until 96 h of glutamine deprivation. Glutamine deprivation resulted in oxidative stress where superoxide levels were significantly elevated after 96 h in the MCF-7-and MDA-MB-231 cell lines. Time-dependent production of hydrogen peroxide was accompanied by aberrant mitochondrial membrane potential. The effects of ROS and mitochondrial membrane potential were more prominently observed in the MCF-7 cell line when compared to the MDA-MB-231-, MCF-10A- and BT-20 cell lines. Cell cycle progression revealed that glutamine deprivation resulted in a significant increase in the S-phase after 72 h of glutamine deprivation in the MCF-7 cell line. Apoptosis induction resulted in a decrease in viable cells in all cell lines following glutamine deprivation. In the MCF-7 cells, 87.61% of viable cells were present after 24 h of glutamine deprivation. CONCLUSION: This study demonstrates that glutamine deprivation resulted in decreased cell proliferation, time-dependent- and cell line-dependent ROS generation, aberrant mitochondrial membrane potential and disrupted cell cycle progression. In addition, the estrogen receptor positive MCF-7 cell line was more prominently affected. This study contributes to knowledge regarding the sensitivity of breast cancer cells and non-tumorigenic cells to glutamine deprivation.

A novel non-sulphamoylated 2-methoxyestradiol derivative causes detachment of breast cancer cells by rapid disassembly of focal adhesions.

BACKGROUND: 2-Methoxyestradiol (2ME2) is an estradiol metabolite with well documented antiproliferative properties in many cancer cell lines. However, it is rapidly metabolised in vivo which limits its clinical application. Therefore, more stable derivatives with potentially improved clinical features have been designed by our group. Here we describe an estrone-like derivative of 2ME2, namely EE-15-one, that unlike other derivatives which induce cell cycle arrest, induces a rapid loss of cell-substrate adhesion through the inactivation and disassembly of focal adhesions. METHODS: To assess the effect of 2-ethyl-estra-1,3,5 (10),15-tetraen-3-ol-17-one (EE-15-one) on breast cancer cell lines, cell survival was quantified. The effect of EE-15-one on cell attachment was assessed by measuring cell adhesion and cell rounding via light microscopy. Effects on focal adhesion dynamics and actin cytoskeleton organisation were visualised by immunofluorescence while focal adhesion signalling was assessed by western blot. Cell death was quantified using a lactate dehydrogenase activity (LDH) assay. To investigate specificity towards cell-substrate over cell-cell contact inhibition, EE-15-one effects on 3D cell cultures were assessed. RESULTS: Cell survival assays show an almost complete loss of cells within 24 h of EE-15-one exposure in contrast to published sulphamoylated 2ME2 derivatives. Cell loss is linked to rapid detachment and adhesion inhibition. Focal adhesion size and number are rapidly diminished while actin fibres became severed and disappeared within 2 h post exposure. These changes were not due to cell necrosis as LDH activity only slightly increased after 24 h. Cells grown in cell-cell adhesion dependent spheroids did not respond to EE-15-one exposure suggesting that EE-15-one specifically inhibits cell-substrate adhesions but not cell-cell adhesions and does not directly impact the actin cytoskeleton. CONCLUSION: We show that a novel 2ME2 derivative, EE-15-one, induces rapid loss of focal adhesion function leading to cell-substrate detachment through interference with integrin-based cell-substrate adhesions, but not cadherin dependent cell-cell adhesions. Therefore, EE-15-one is the first 2ME2 derivative that has an alternative mode of action to the antimitotic activity of 2ME2. As such EE-15-one shows potential as a lead compound for further development as an inhibitor of cell-substrate adhesion which is essential for metastatic dissemination.

Promising anticancer activity of batumin: a natural polyene antibiotic produced by Pseudomonas batumici.

AIM: To determine the computer-predicted anticancer activity of antibiotic batumin. MATERIALS & METHODS: Cytotoxicity assays, cell morphology microscopy and cell cycle progression were studied in cancer and nontumorigenic cell lines. An in vivo experiment on Lewis lung carcinoma (3LL)-transplanted mice was conducted to evaluate potential antimetastatic. RESULTS & CONCLUSION: Cytotoxicity against melanoma and lung carcinoma cells (IC50 ≈ 5 µg/ml) was detected. Hypercondensed chromatin and apoptotic body formation in batumin-treated cells suggested the induction of apoptosis supported also by an observed increase in the quantity of cells occupying the sub-G1 cell cycle phase. Twofold reduction in the number and volume of lung metastases in Lewis lung carcinoma (3LL)-bearing batumin-treated mice was demonstrated. Highly specific cytotoxicity of batumin against cancer cell lines potentiates further studies.

Crosstalk between the Warburg effect, redox regulation and autophagy induction in tumourigenesis.

Tumourigenic tissue uses modified metabolic signalling pathways in order to support hyperproliferation and survival. Cancer-associated aerobic glycolysis resulting in lactic acid production was described nearly 100 years ago. Furthermore, increased reactive oxygen species (ROS) and lactate quantities increase metabolic, survival and proliferation signalling, resulting in increased tumourigenesis. In order to maintain redox balance, the cell possesses innate antioxidant defence systems such as superoxide dismutase, catalase and glutathione. Several stimuli including cells deprived of nutrients or failure of antioxidant systems result in oxidative stress and cell death induction. Among the cell death machinery is autophagy, a compensatory mechanism whereby energy is produced from damaged and/or redundant organelles and proteins, which prevents the accumulation of waste products, thereby maintaining homeostasis. Furthermore, autophagy is maintained by several pathways including phosphoinositol 3 kinases, the mitogen-activated protein kinase family, hypoxia-inducible factor, avian myelocytomatosis viral oncogene homolog and protein kinase receptor-like endoplasmic reticulum kinase. The persistent potential of cancer metabolism, redox regulation and the crosstalk with autophagy in scientific investigation pertains to its ability to uncover essential aspects of tumourigenic transformation. This may result in clinical translational possibilities to exploit tumourigenic oxidative status and autophagy to advance our capabilities to diagnose, monitor and treat cancer.

A bis-sulphamoylated estradiol derivative induces ROS-dependent cell cycle abnormalities and subsequent apoptosis.

Clinical trials have revealed that the potential anticancer agent, 2-methoxyestradiol (2ME2) has limitations due to its low bioavailability. Subsequently, 2ME2 derivatives including (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) have shown improved efficacies in inducing apoptosis. However, no conclusive data exist to explain the mode of action exerted by these drugs. This study investigated the mode of action used by EMBS as a representative of the sulphamoylated 2ME2 derivatives. Hydrogen peroxide and superoxide production was quantified using dichlorofluorescein diacetate and hydroethidine. Cell proliferation and mitochondrial metabolism were investigated using crystal violet and Alamar Blue. Apoptosis was assessed using Annexin V-FITC while mitochondrial integrity was assessed using Mitocapture. Autophagy was visualised using LC3B II antibodies. The effects of EMBS on H2A phosphorylation and nuclei were visualised using phospho H2A antibody and 4',6-diamidino-2-phenylindole, dihydrochloride. Data showed that EMBS exposure leads to increased reactive oxygen species (ROS) production which is correlated with loss of cell proliferation, mitochondrial membrane damage, decreased metabolic activity, G2/M arrest, endoreduplication, DNA double stranded breaks, micronuclei and apoptosis induction. Treatment of EMBS-exposed cells with the ROS scavenger, N-acetyl cysteine, abrogated ROS production, cell cycle arrest and apoptosis implying an essential role for ROS production in EMBS signaling. The inhibition of c-Jun N-terminal kinase (JNK) activity also inhibited EMBS-induced apoptosis suggesting that EMBS triggers apoptosis via the JNK pathway. Lastly, evaluation of LC3IIB protein levels indicated that autophagy is not activated in EMBS-exposed cells. Our data shows that EMBS targets a pathway that leads to increased ROS production as an early event that culminates in G2/M arrest and apoptosis by means of JNK-signaling in cancer cells. This study suggests a novel oxidative stress-dependent mode of action for sulphamoylated derivatives.

In vitro assessment of a computer-designed potential anticancer agent in cervical cancer cells.

BACKGROUND: Computer-based technology is becoming increasingly essential in biological research where drug discovery programs start with the identification of suitable drug targets. 2-Methoxyestradiol (2ME2) is a 17ß-estradiol metabolite that induces apoptosis in various cancer cell lines including cervical cancer, breast cancer and multiple myeloma. Owing to 2ME2's poor in vivo bioavailability, our laboratory in silico-designed and subsequently synthesized a novel 2ME2 analogue, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol), using receptor- and ligand molecular modeling. In this study, the biological effects of ESE-15-ol (180 nM) and its parent molecule, 2ME2 (1 µM), were assessed on morphology and apoptosis induction in cervical cancer cells. RESULTS: Transmission electron microscopy, scanning electron microscopy and polarization-optical transmitted light differential interference contrast (PlasDIC) images demonstrated morphological hallmarks of apoptosis including apoptotic bodies, shrunken cells, vacuoles, reduced cell density and cell debris. Flow cytometry analysis showed apoptosis induction by means of annexin V-FITC staining. Cell cycle analysis showed that ESE-15-ol exposure resulted in a statistically significant increase in the G2M phase (72%) compared to 2ME2 (19%). Apoptosis induction was more pronounced when cells were exposed to ESE-15-ol compared to 2ME2. Spectrophotometric analysis of caspase 8 activity demonstrated that 2ME2 and ESE-15-ol both induced caspase 8 activation by 2- and 1.7-fold respectively indicating the induction of the apoptosis. However, ESE-15-ol exerted all of the above-mentioned effects at a much lower pharmacological concentration (180 nM) compared to 2ME2 (1 µM physiological concentration). CONCLUSION: Computer-based technology is essential in drug discovery and together with in vitro studies for the evaluation of these in silico-designed compounds, drug development can be improved to be cost effective and time consuming. This study evaluated the anticancer potential of ESE-15-ol, an in silico-designed compound in vitro. Research demonstrated that ESE-15-ol exerts antiproliferative activity accompanied with apoptosis induction at a nanomolar concentration compared to the micromolar range required by 2ME2. This study is the first study to demonstrate the influence of ESE-15-ol on morphology, cell cycle progression and apoptosis induction in HeLa cells. In silico-design by means of receptor- and ligand molecular modeling is thus effective in improving compound bioavailability while preserving apoptotic activity in vitro.

In vitro assessment of a computer-designed potential anticancer agent in cervical cancer cells

BACKGROUND: Computer-based technology is becoming increasingly essential in biological research where drug discovery programs start with the identification of suitable drug targets. 2-Methoxyestradiol (2ME2) is a 17ß-estradiol metabolite that induces apoptosis in various cancer cell lines including cervical cancer, breast cancer and multiple myeloma. Owing to 2ME2's poor in vivo bioavailability, our laboratory in silico-designed and subsequently synthesized a novel 2ME2 analogue, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol), using receptor- and ligand molecular modeling. In this study, the biological effects of ESE-15-ol (180 nM) and its parent molecule, 2ME2 (1 µM), were assessed on morphology and apoptosis induction in cervical cancer cells. RESULTS: Transmission electron microscopy, scanning electron microscopy and polarization-optical transmitted light differential interference contrast (PlasDIC) images demonstrated morphological hallmarks of apoptosis including apoptotic bodies, shrunken cells, vacuoles, reduced cell density and cell debris. Flow cytometry analysis showed apoptosis induction by means of annexin V-FITC staining. Cell cycle analysis showed that ESE-15-ol exposure resulted in a statistically significant increase in the G2M phase (72%) compared to 2ME2 (19%). Apoptosis induction was more pronounced when cells were exposed to ESE-15-ol compared to 2ME2. Spectrophotometric analysis of caspase 8 activity demonstrated that 2ME2 and ESE-15-ol both induced caspase 8 activation by 2- and 1.7-fold respectively indicating the induction of the apoptosis. However, ESE-15-ol exerted all of the above-mentioned effects at a much lower pharmacological concentration (180 nM) compared to 2ME2 (1 µM physiological concentration). CONCLUSION: Computer-based technology is essential in drug discovery and together with in vitro studies for the evaluation of these in silico-designed compounds, drug development can be improved to be cost effective and time consuming. This study evaluated the anticancer potential of ESE-15-ol, an in silico-designed compound in vitro. Research demonstrated that ESE-15-ol exerts antiproliferative activity accompanied with apoptosis induction at a nanomolar concentration compared to the micromolar range required by 2ME2. This study is the first study to demonstrate the influence of ESE-15-ol on morphology, cell cycle progression and apoptosis induction in HeLa cells. In silico-design by means of receptor- and ligand molecular modeling is thus effective in improving compound bioavailability while preserving apoptotic activity in vitro.

Antimitotic drugs in the treatment of cancer.

Cancer is a complex disease since it is adaptive in such a way that it can promote proliferation and invasion by means of an overactive cell cycle and in turn cellular division which is targeted by antimitotic drugs that are highly validated chemotherapy agents. However, antimitotic drug cytotoxicity to non-tumorigenic cells and multiple cancer resistance developed in response to drugs such as taxanes and vinca alkaloids are obstacles faced in both the clinical and basic research field to date. In this review, the classes of antimitotic compounds, their mechanisms of action and cancer cell resistance to chemotherapy and other limitations of current antimitotic compounds are highlighted, as well as the potential of novel 17-ß estradiol analogs as cancer treatment.

Influence of partial and complete glutamine-and glucose deprivation of breast-and cervical tumorigenic cell lines.

BACKGROUND: Due to their high proliferative requirements, tumorigenic cells possess altered metabolic systems whereby cells utilize higher quantities of glutamine and glucose. These altered metabolic requirements make it of interest to investigate the effects of physiological non-tumorigenic concentrations of glucose and glutamine on tumorigenic cells since deprivation of either results in a canonical amino acid response in mammalian cell. METHODS: The influence of short-term exposure of tumorigenic cells to correlating decreasing glutamine- and glucose quantities were demonstrated in a highly glycolytic metastatic breast cell line and a cervical carcinoma cell line. Thereafter, cells were propagated in medium containing typical physiological concentrations of 1 mM glutamine and 6 mM glucose for 7 days. The effects on morphology were investigated by means of polarization-optical transmitted light differential interference contrast. Flow cytometry was used to demonstrate the effects of glutamine-and glucose starvation on cell cycle progression and apoptosis induction. Fluorometrics were also conducted to investigate the effects on intrinsic apoptosis induction (mitocapture), reactive oxygen species production (2,7-dichlorofluorescein diacetate) and acidic vesicle formation (acridine orange). RESULTS: Morphological data suggests that glutamine-and glucose deprivation resulted in reduced cell density and rounded cells. Glutamine-and glucose starvation also resulted in an increase in the G2M phase and a sub-G1 peak. Complete starvation of glutamine and glucose resulted in the reduction of the mitochondrial membrane potential in both cell lines with MDA-MB-231 cells more prominently affected when compared to HeLa cells. Further, starved cells could not be rescued sufficiently by propagating since cells possessed an increase in reactive oxygen species, acidic compartments and vacuole formation. CONCLUSION: Starvation from glutamine and glucose for short periods resulted in decreased cell density, rounded cells and apoptosis induction by means of reactive oxygen species generation and mitochondrial dysfunction. In addition, the metastatic cell line reacted more prominently to glutamine-and glucose starvation due to their highly glycolytic nature. Satisfactory cellular rescue was not possible as cells demonstrated oxidative stress and depolarized mitochondrial membrane potential. This study contributes to the knowledge regarding the in vitro effects and signal transduction of glucose and/or l-glutamine deprivation in tumorigenic cell lines.

A 2-methoxyestradiol bis-sulphamoylated derivative induces apoptosis in breast cell lines.

INTRODUCTION: Research involving antimitotic compounds identified 2-methoxyestradiol (2ME2), as a promising anticancer endogenous metabolite. Owing to its low bioavailability, several in silico-designed 2ME2 analogues were synthesized. Structure-activity relationship studies indicated that an already existing 17-ß-estradiol analogue, namely (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) to exert potential in vitro anticancer activity. METHODS: This study investigated the in vitro apoptotic influence of EMBS in an estrogen receptor-positive breast adenocarcinoma epithelial cell line (MCF-7); an estrogen receptor-negative breast epithelial cell line (MDA-MB-231) and a non-tumorigenic breast cell line (MCF-12A). Cell cycle progression, a phosphatidylserine flip, caspase 6-, 7- and 8 enzyme activity levels, Bcl-2 phosphorylation status at serine 70 and Bcl-2- and p53 protein levels were investigated to identify a possible action mechanism for apoptotic induction. RESULTS: The xCELLigence real-time label-independent approach revealed that EMBS exerted antiproliferative activity in all three cell lines after 24 h of exposure. A G2M block was observed and apoptosis induction was verified by means of flow cytometry using propidium iodide and Annexin V-FITC respectively. EMBS-treated cells demonstrated a reduced mitochondrial membrane potential. EMBS exposure resulted in a statistically significant increase in p53 protein expression, decreased Bcl-2 protein expression and a decrease in pBcl-2(s70) phosphorylation status in all three cell lines. Results support the notion that EMBS induces apoptosis in all three cell lines. CONCLUSION: This study includes investigation into the apoptotic hallmarks exerted by EMBS after exposure of three cell lines namely MCF-7-, MDA-MDA-231- and MCF-12A cells. Increased caspase 6-, caspase 7- and caspase 8 activities, upregulation of p53 protein expression and a decrease in phosphorylation status of Bcl-2 at serine 70 in tumorigenic and non-tumorigenic lines were demonstrated.