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.

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.

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.

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.

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.

In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17-ß-estradiol analogue in breast adenocarcinoma cells.

2-Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low oral bioavailability, several promising analogues of 2-methoxyestradiol have been developed. In this study, the in vitro influence of the compound, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19), a non-commercially available 17-ß-estradiol analogue, was tested on the breast adenocarcinoma MCF-7 cell line. The in vitro influence of 24 h exposure to 0.18 µM of C19 on MCF-7 cells was evaluated on cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization-optical transmitted light differential interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and compromised cell density in C19-treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes, were also observed in C19-treated samples. An increase in the number of cells present in the sub-G1 fraction, as well as a reduction in mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in aggresome formation was observed in C19-treated cells. C19 induced both apoptosis and autophagy in MCF-7 cells.

2-Methoxyestradiol-bis-sulphamate refrains from inducing apoptosis and autophagy in a non-tumorigenic breast cell line.

BACKGROUND: Anticancer research resulted in the discovery of a promising antimitotic metabolite, 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate, a bis-sulphamoylated analogue exerts antiproliferative- and antimitotic activity. Investigating the anticancer potential of 2-methoxyestradiol-bis-sulphamate requires demonstrating the influence of 2-methoxyestradiol-bis-sulphamate on non-tumorigenic cells. This project focused on the in vitro effects of 2-methoxyestradiol-bis-sulphamate on the non-tumorigenic MCF-12A breast epithelial cell line. METHODS: The in vitro influence of 2-methoxyestradiol-bis-sulphamate was investigated on cell cycle progression, possible induction of apoptosis and autophagy and reactive oxygen species generation. Cell cycle progression was done using flow cytometry in conjunction with ethanol fixation and propidium iodide staining. Displaying effects on the mitochondrial membrane potential was achieved utilizing flow cytometry and the MitoCapture TM Mitochondrial apoptosis detection kit. Autophagy detection was done by means of flow cytometry and anti-LC3B conjugated to DyLight 488. Reactive oxygen species generation was conducted employing flow cytometry and 2,7-dichlorofluorescein diacetate and hydroethidine. RESULTS: This study demonstrated that 2-methoxyestradiol-bis-sulphamate did not affect cell cycle progression or reactive oxygen species in a statistically significant manner in the non-tumorigenic MCF-12A cell line. In addition, 2-methoxyestradiol-bis-sulphamate did not statistically significantly induce apoptosis or autophagy. CONCLUSION: Reports indicate that 2-methoxyestradiol-bis-sulphamate induces apoptosis and autophagy in several tumorigenic cell lines. The anticancer ability of 2-methoxyestradiol-bis-sulphamate is due to its antimitotic activity. However, this study demonstrates the promising notion that 2-methoxyestradiol-bis-sulphamate does not affect the non-tumorigenic MCF-12A cells. This project contributes to the embedded scientific knowledge regarding the differential death mechanisms used by 2-methoxyestradiol-bis-sulphamate on tumorigenic and non-tumorigenic cell lines.

In vitro effects of 2-methoxyestradiol-bis-sulphamate on reactive oxygen species and possible apoptosis induction in a breast adenocarcinoma cell line.

BACKGROUND: In the search for anticancer agents, a promising 17-ß-estradiol metabolite, 2-methoxyestradiol (2ME2) was found that exerts antiproliferative in vitro and in vivo activity. Since 2ME2 has limited biological accessibility and rapid metabolic degradation, the purpose of this study was to investigate the in vitro influence exerted by an analogue of 2ME2 namely 2-methoxyestradiol-bis-sulphamate (2MEBM) in a breast adenocarcinoma cell line (MCF-7). METHODS: This was conducted by investigating 2MEBM's in vitro influence on cell cycle progression, mitochondrial membrane potential and possible production of reactive oxygen species (ROS) generation. In vitro effects of 2MEBM on cell cycle progression was demonstrated by means of flow cytometry using propidium iodide. Hydrogen peroxide and superoxide production was investigated using 2,7-dichlorofluorescein diacetate and hydroethidine, respectively. The probable reduction in the mitochondrial membrane potential was demonstrated using a MitoCapture™ kit. RESULTS: Cell cycle progression revealed the presence of a sub-G1 apoptotic peak. Reduction of mitochondrial membrane potential after exposure to 2MEBM was demonstrated and an increase in ROS production was also observed. CONCLUSION: This study verified that 2MEBM exposure resulted in apoptosis induction, increased ROS production and reduced mitochondrial membrane potential in a tumorigenic breast epithelial cell line. Data obtained from this project contributes to the unravelling of the in vitro signal transduction of 2MEBM in tumorigenic cell lines.

The in vitro effects of 2-methoxyestradiol-bis-sulphamate on cell numbers, membrane integrity and cell morphology, and the possible induction of apoptosis and autophagy in a non-tumorigenic breast epithelial cell line.

2-methoxyestradiol (2ME2) exerts estrogen receptor-independent anti-proliferative, anti-angiogenic and anti-tumor activity in vitro and in vivo. Due to its low bioavailability and rapid metabolic degradation, several analogues have been developed in recent years. 2-methoxyestradiol-bis-sulphamate (2-MeOE2bisMATE) is a bis-sulphamoylated derivative of 2ME2 with anti-proliferative activity. The aim of this study was to investigate cell signaling events induced by 2-MeOE2bisMATE in a non-tumorigenic cell line (MCF-12A) by analysing its influence on cell number, morphology and membrane integrity, and the possible induction of apoptosis and autophagy. Dose- and time-dependent studies revealed that 48 h exposure to 2-MeOE2bisMATE (0.4 µM) resulted in a decrease in cell numbers to 79%. A slight increase in the level of lactate dehydrogenase production was observed in the 2-MeOE2bisMATE-treated cells. Morphological studies revealed an increase in the number of cells in metaphase. Hallmarks of apoptosis were also found, namely nuclear fragmentation and apoptotic bodies. In addition, increased lysosomal staining was observed via fluorescent microscopy, suggesting the induction of another type of cell death, namely autophagy. Since 2-MeOE2bisMATE is regarded as a potential anti-cancer agent, it is also imperative to investigate the susceptibility of non-tumorigenic cells to its influence. The data generated from this study contributes to the understanding of the action that 2-MeOE2bisMATE exerts on the non-tumorigenic MCF-12A breast epithelial cell line.