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Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost-effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. We describe a bead-based screening assay for S1-neutralization using recombinant fluorescent proteins of hACE2 and SARS-CoV2-S1, immobilized on solid beads employing nanobodies/metal-affinity tags. Nanobody-mediated capture of SARS-CoV-2-Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single-color fluorescent imaging of ACE2-GFPSpark binding to His-tagged S1-Receptor Binding Domain (RBD-His) immobilized beads. The second approach is dual-color imaging of soluble ACE2-GFPSpark to S1-Orange Fluorescent Protein (S1-OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening.

Cancer cells grown as 3D-structures are better models for mimicking in vivo conditions than the 2D-culture systems employable in drug discovery applications. Cell cycle and cell death are important determinants for preclinical drug screening and tumor growth studies in laboratory conditions. Though several 3D-models and live-cell compatible approaches are available, a method for simultaneous real-time detection of cell cycle and cell death is required. Here we demonstrate a high-throughput adaptable method using genetically encoded fluorescent probes for the real-time quantitative detection of cell death and cell cycle. The cell-cycle indicator cdt1-Kusabira orange (KO) is stably integrated into cancer cells and further transfected with the Fluorescence Resonance Energy Transfer-based ECFP-DEVD-EYFP caspase activation sensor. The nuclear cdt1-KO expression serves as the readout for cell-cycle, and caspase activation is visualized by ECFP/EYFP ratiometric imaging. The image-based platform allowed imaging of growing spheres for prolonged periods in 3D-culture with excellent single-cell resolution through confocal microscopy. High-throughput screening (HTS) adaptation was achieved by targeting the caspase-sensor at the nucleus, which enabled the quantitation of cell death in 3D-models. The HTS using limited compound libraries, identified two lead compounds that induced caspase-activation both in 2D and 3D-cultures. This is the first report of an approach for noninvasive stain-free quantitative imaging of cell death and cell cycle with potential drug discovery applications.

The utilization of peptide-based drug delivery systems has been suboptimal due to their poor proteolytic susceptibility, poor cell permeability, and limited tumor homing capabilities. Earlier attempts in using d-enantiomers in peptide sequences increased proteolytic stability but have compromised the overall penetration capability. We designed a series of peptides (STRAPs) with a syndiotactic polypeptide backbone that can potentially form a spatial array of cationic groups, an important feature that facilitates cellular uptake. The peptides penetrate cell membranes through a combination of active and passive modes. Furthermore, the cellular uptake of the peptides was unaffected by the presence of or treatment with bovine serum and human plasma. The designed peptides successfully delivered methotrexate, an anticancer drug, to the

Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost-effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. Here, we describe a bead-based screening assay for S1-neutralization using recombinant fluorescent proteins of hACE2 and SARS-CoV2-S1, immobilized on solid beads employing nanobodies /metal-affinity tags. Nanobody-mediated capture of SARS-CoV-2 - Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single-color fluorescent imaging of ACE2–GFPspark binding to His-tagged S1-Receptor Binding Domain (RBD-His) immobilized beads. The second approach is dual-color imaging of soluble ACE2-GFPSpark to S1-Orange Fluorescent Protein (S1-OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening. Life-time imaging of the ACE2-GFPSpark confirmed the interaction of ACE2 and S1-OFPSpark on beads. The self-renewable source of secreted recombinant proteins from stable cells and its direct use without necessitating purification renders the platform a cost-effective and rapid one than the popular pseudovirion assay and live virus-based assays. Any laboratory with minimum expertise can rapidly perform this bead assay for neutralizing antibody detection using stable engineered cells.

Most toxic compounds including cancer drugs target mitochondria culminating in its permeabilization. Cancer drug-screening and toxicological testing of compounds require cost-effective and sensitive high-throughput methods to detect mitochondrial damage. Real-time methods for detection of mitochondrial damage are less toxic, allow kinetic measurements with good spatial resolution and are preferred over end-stage assays. Cancer cell lines stably expressing genetically encoded mitochondrial-targeted redox-GFP2 (mt-roGFP) were developed and validated for its suitability as a mitochondrial damage sensor. Diverse imaging platforms and flow-cytometry were utilized for ratiometric analysis of redox changes with known toxic and cancer drugs. Key events of cell death and mitochondrial damage were studied at single-cell level coupled with mt-roGFP. Cells stably expressing mt-roGFP and H2B-mCherry were developed for high-throughput screening (HTS) application. Most cancer drugs while inducing mitochondrial permeabilization trigger mitochondrial-oxidation that can be detected at single-cell level with mt-roGFP. The image-based assay using mt-roGFP outperformed other quantitative methods of apoptosis in ease of screening. Incorporation of H2B-mCherry ensures accurate and complete automated segmentation with excellent Z value. The results substantiate that most cancer drugs and known plant-derived antioxidants trigger cell-death through mitochondrial redox alterations with pronounced ratio change in the mt-roGFP probe. Real-time analysis of mitochondrial oxidation and mitochondrial permeabilization reveal a biphasic ratio change in dying cells, with an initial redox surge before mitochondrial permeabilization followed by a drastic increase in ratio after complete mitochondrial permeabilization. Overall, the results prove that mitochondrial oxidation is a reliable indicator of mitochondrial damage, which can be readily determined in live cells using mt-roGFP employing diverse imaging techniques. The assay described is highly sensitive, easy to adapt to HTS platforms and is a valuable resource for identifying cytotoxic agents that target mitochondria and also for dissecting cell signaling events relevant to redox biology., Highlights • Mitochondrial oxidation is an universal marker for mitochondrial damage and mitochondrial permeabilization. • Ratiometric imaging of mt-roGFP in high-throughput mode allows rapid screening of compounds that target mitochondria. • Real-time ratiometric imaging of mt-roGFP and mitochondrial permeabilization reveals a biphasic redox alteration in cells undergoing mitochondrial permeabilization.

Cancer growth and progression are associated with immune suppression. Cancer cells have the ability to activate different immune checkpoint pathways that harbor immunosuppressive functions. Monoclonal antibodies that target immune checkpoints provided an immense breakthrough in cancer therapeutics. Among the immune checkpoint inhibitors, PD-1/PD-L1 and CTLA-4 inhibitors showed promising therapeutic outcomes, and some have been approved for certain cancer treatments, while others are under clinical trials. Recent reports have shown that patients with various malignancies benefit from immune checkpoint inhibitor treatment. However, mainstream initiation of immune checkpoint therapy to treat cancers is obstructed by the low response rate and immune-related adverse events in some cancer patients. This has given rise to the need for developing sets of biomarkers that predict the response to immune checkpoint blockade and immune-related adverse events. In this review, we discuss different predictive biomarkers for anti-PD-1/PD-L1 and anti-CTLA-4 inhibitors, including immune cells, PD-L1 overexpression, neoantigens, and genetic and epigenetic signatures. Potential approaches for further developing highly reliable predictive biomarkers should facilitate patient selection for and decision-making related to immune checkpoint inhibitor-based therapies., Cancer immunotherapy: Markers of success Biomarkers that predict a patient’s response to cancer immunotherapy are being developed, and may allow individual tailoring of therapies. Cancer cells can suppress the immune system by activating immune checkpoints, signals that stop the immune system from attacking the host. Immunotherapy, a recently developed treatment, inactivates immune checkpoints, restoring the patient’s immune response against the tumor. Although promising, immunotherapy has a low response rate, and sometimes triggers severe auto-immune reactions. Eyad Elkord at Qatar Biomedical Research Institute in Qatar and coworkers have reviewed biomarkers that could be used to predict individual effects of immunotherapy, identifying nonresponders and preventing adverse immune reactions. They discuss markers based on factors such as immune response level and individual tumor genetics, among others. With further research and testing, these biomarkers could improve the efficacy of this promising new cancer therapy.

Ketogenesis is the production of ketone bodies, which provide energy when the body lacks glucose. Under ketogenic conditions, the body switches from primarily carbohydrate to fat metabolism to maintain energy balance. However, accumulation of high levels of ketone bodies in the blood results in ketosis. Treating ketosis with natural substances is preferable, because they are unlikely to cause side-effects. Momilactone B is an active compound isolated from Korean rice. Based on previous studies, we hypothesized that momilactone B could inhibit ketosis. We constructed an in vitro ketosis model by glucose starvation. We used this model to test the anti-ketosis effects of momilactone B. A primary target for treating ketosis is angiopoietin-like-3 (ANGPTL3), which modulates lipoprotein metabolism by inhibiting lipoprotein lipase (LPL), a multifunctional enzyme that breaks down stored fat to produce triglycerides. We showed that momilactone B could regulate the ANGPTL3-LPL pathway. However, a strong anti-ketosis candidate drug should also inhibit ketogenesis. Ketogenesis can be suppressed by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2), a mitochondrial enzyme that converts acetyl-CoA to ketone bodies. We found that momilactone B suppressed the expression of HMGCS2 through the increased expression of STAT5b. We also elucidated the relationship of STAT5b to ANGPTL3 and LPL expression.

The development of the smart drug delivery system (SDDS) is a revolution of nanotechnology in the pharmaceutical and medical fields. Nanomaterials through their enhanced target specificity, biodistribution, and plasma retention have overcome the therapeutic adverse effects of the conventional drug delivery system. The response-based intelligence of the system helps to release drug payloads released under specific pathological conditions. The development of an intelligent drug delivery system combines multiple approaches and multiple signal responses to improve their applicability in diagnosis as well as therapy. This chapter provides a brief overview of the SDDSs with respect to various stimuli-responsive systems.

As human lifespans have increased, the incidence of osteoporosis has also increased. Methylsulfonylmethane (MSM) affects the process of mesenchymal stem cell (MSC) differentiation into osteoblasts via the Janus kinase 2 (Jak2)/signal transducer and activator of transcription (STAT)5b signaling pathway, and bone morphogenetic protein 2 (BMP‑2) is also known to significantly affect bone health. In addition, the phosphorylation of small mothers against decapentaplegic (Smad)1/5/8 regulates the Runt‑related transcription factor 2 (Runx2) gene, which encodes a transcription factor for osteoblast differentiation markers. In the present study, the differentiation of MSCs treated with MSM, BMP‑2, and their combination were examined. The differentiation of osteoblasts was demonstrated through observation of morphological changes and mineralization, using alizarin red and Von Kossa staining. Western blotting analysis demonstrated that the combination of MSM and BMP-2 increased the phosphorylation of the BMP signaling-associated protein, Smad1/5/8. Combination of MSM and BMP-2 significantly increased osteogenic differentiation and mineralization of the MSCs compared with either MSM or BMP-2 alone. Additionally, reverse transcription-polymerase chain reaction analysis demonstrated that combination of MSM and BMP-2 increased the expression level of the Runx2 gene and the osteoblast differentiation marker genes, alkaline phosphatase, bone sialoprotein and osteocalcin, in MSCs compared with controls. Thus, the combination of MSM and BMP-2 may promote the differentiation of MSCs into osteoblasts.

Breast cancer is the most common cancer in women globally. The factors that increase risk include: late age at first birth, alcohol, radiation exposure, family history of breast cancer, and postmenopausal hormone therapy. Numerous drugs are being developed to treat breast cancer. Among them, Herceptin is used for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cases and targets HER2 effectively and efficiently, but it is very expensive. Methylsulfonylmethane (MSM) is an organic sulfur-containing natural compound having no reported toxicity. We examined MSM in breast cancer cell lines and found it inhibited the proliferation of estrogen receptor-positive and HER2-positive breast cancer cells in a dose-dependent manner. It also suppressed the activation of STAT5b and expression of HER2 in breast cancer cells. We determined the STAT5b binding site (GAS element) in the HER2 gene. Detailed analysis showed that MSM decreased the ability of STAT5b to bind the promoter of the HER2 gene and a luciferase assay demonstrated reduced activity. We confirmed that MSM can effectively regulate STAT5b, and thereby decrease HER2 expression. Therefore, we recommend the use of MSM as an inhibitor for the management of HER2-positive breast cancers.

Background Photodynamic therapy (PDT) is a successful cancer treatment modality. In vitro, in vivo, and clinical studies with different photosensitizers reveal diverging cell fates, including apoptosis, necrosis, autophagy, and non-specific forms of cell death. The mode of action and efficacy of PDT is mediated through free radical generation and is highly dependent on diverse variables such as nature, dose, metabolism of photosensitizer, irradiation energy, and irradiation cycle. Aim Discovery of newer photosensitizers and optimization of PDT approaches to achieve a clinically relevant form of cell death called apoptosis requires better in vitro real-time methods. Oxidative damage and mitochondrial permeabilization are critical signaling events involved in photodamage and apoptosis. Hence, mitochondrial damage detection is an appropriate target signaling for mechanistic evaluation of PDT. Methodology We report mitochondria-targeted redox GFP expressing cells as a sensitive system to test and validate important variables of PDT using the photosensitizer 5-Aminolevulinic acid (5-ALA) as a model. An independent FRET-based caspase sensor cell was also used to study the impact of the photosensitizer dosage and irradiation duration on the mode of cell death. Results The study reveals that the cancer cells expressing mt-roGFP are extremely sensitive to monitor mitochondrial oxidation induced by PDT. The extent of mitochondrial redox changes induced by PDT can be determined using these sensor cells by real-time image-based approaches. These approaches provide sufficient temporal resolution that is required to fine-tune and optimize the PDT conditions. The degree of oxidation of the probe is highly dependent on the dosage of photosensitizer and duration of light irradiation, which determines the nature of cell death. A real-time caspase sensor probe further confirmed that the caspase-dependent and caspase-independent nature of cell death is in high correlation with the extent of mitochondrial oxidation. A condition that triggers rapid and extreme mito-oxidation seems to favor necrosis, while delayed and slowly progressing redox changes contribute to caspase-dependent apoptosis. Conclusion The study confirms that temporal analysis of mitochondrial oxidation is a reliable biomarker for fine-tuning PDT conditions to achieve the desired outcome. This can be achieved using stable cancer cell lines expressing mitochondria-targeted roGFP by ratiometric imaging.

Targeted cancer therapy with natural compounds is more effective than nontargeted therapy. Nobiletin is a flavonoid derived from citrus peel that has anticancer activity. Cluster of differentiation 36 (CD36) is a member of the class B scavenger receptor family that is involved in importing fatty acids into cells. CD36 plays a role in tumor angiogenesis by binding to its ligand, thrombospondin-1 (TSP-1), and then interacting with transforming growth factor beta 1 (TGFβ1). CD36 is implicated in tumor metastasis through its roles in fatty acid metabolism. This study investigated the molecular mechanisms underlying nobiletin’s anticancer activity by characterizing its interactions with CD36 as the target molecule. We hypothesize that the anti-angiogenic activity of nobiletin involving its regulation of CD36 via signal transducer and activator of transcription 3 (STAT3) rather than through TSP-1. Gene analysis identified a Gamma interferon activation site (GAS) element in the CD36 gene promoter that acts as a STAT3 binding site, an interaction that was confirmed by ChIP assay. STAT3 interacts with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), suggesting that nobiletin also acts through the CD36/ (STAT3)/NF-κB signaling axis. Nobiletin inhibited CD36-dependent breast cancer cell migration and invasion as well as CD36-mediated tumor sphere formation. Taken together, these results suggest that nobiletin inhibits cancer stem cells in multiple ways.

Tannic acid (TA), is a potent anti-oxidant, showing anti-proliferative effects on numerous cancers. The ability of TA to induce proliferation inhibition on the rare tumor, gingival squamous cell carcinoma (GSCC), comprising

Sorghum contains phytochemicals that can protect against multiple forms of cancers. However, the underlying molecular targets and cellular mechanisms remain unknown. We investigated the role of Hwanggeumchal sorghum extracts (HSE) on colon cancer cells. We found HSE inhibited the proliferation of human colon cancer cells in a dose-dependent manner by inducing G1 phase arrest and apoptosis. Likewise, it suppressed the Jak2/STAT3 and PI3K/AKT/mTOR pathways. Studies on STAT3 transcription regulatory functions showed HSE inhibited the STAT3/DNA binding and transcription promoter activity. Thereby the expression of STAT3 downstream targets VEGF and VEGF-R2 were dropped. Furthermore, HSE treatment suppressed tumor growth and pulmonary metastasis in animal models. Together, these findings suggested that the targeted inhibition of Jak2/STAT3 and PI3K/Akt/mTOR contribute to cancer therapy potential of H. sorghum (HS). Thus, use of sorghum as a dietary supplement has the potential as a nutraceutical to prevent the onset of colon cancer, without any side effects.

Ketosis is a condition where ketone bodies are used as the energy source instead of glucose. The state of ketosis becomes dangerous if the fat metabolism in the body is high. When the glucose availability gets limited, liver activates fat metabolism to make glucose for energy. As the results of these, ketone bodies will form resulting the state of ketosis. In the present study, we analyzed the in vitro ketosis condition and the effect of glucose level in ketosis induction. The natural organic sulfur containing compound methylsulfonylmethane (MSM) was analyzed for anti-ketosis activity in normal mouse hepatocyte, FL83B cell model system. Ketosis condition was induced by modulating glucose concentration. Growth hormone receptor (GHR) has a great role in the ketosis condition. We hypothesize that MSM has the ability to regulate GHR signaling through signal transducer and activator of transcription 5b (STAT5b). The whole cell lysate analysis and DNA binding activity analysis also suggested the critical role o...

Gingival squamous cell carcinoma is a rare form of cancer that accounts for less than 10% of all head and neck cancers. Targeted therapies with natural compounds are of interest because they possess high efficacy with fewer side-effects. Methylsulfonylmethane (MSM) is an organic sulfur-containing compound with anticancer activities. The main goal of this study was to induce proliferation inhibition and apoptosis in the metastatic YD-38 cell line. MSM up-regulated expression of P21

Tannic acid (TA), a naturally occurring polyphenol, is a potent anti‐oxidant with anti‐proliferative effects on multiple cancers. However, its ability to modulate gene‐specific expression of tumour suppressor genes and oncogenes has not been assessed. This work investigates the mechanism of TA to regulate canonical and non‐canonical STAT pathways to impose the gene‐specific induction of G1‐arrest and apoptosis. Regardless of the p53 status and membrane receptors, TA induced G1‐arrest and apoptosis in breast cancer cells. Tannic acid distinctly modulated both canonical and non‐canonical STAT pathways, each with a specific role in TA‐induced anti‐cancer effects. Tannic acid enhanced STAT1 ser727 phosphorylation via upstream serine kinase p38. This STAT1 ser727 phosphorylation enhanced the DNA‐binding activity of STAT1 and in turn enhanced expression of p21Waf1/Cip1. However, TA binds to EGF‐R and inhibits the tyrosine phosphorylation of both STAT1 and STAT3. This inhibition leads to the inhibition of STAT3/BCL‐2 DNA‐binding activity. As a result, the expression and mitochondrial localization of BCl‐2 are declined. This altered expression and localization of mitochondrial anti‐pore factors resulted in the release of cytochrome c and the activation of intrinsic apoptosis cascade involving caspases. Taken together, our results suggest that TA modulates EGF‐R/Jak2/STAT1/3 and P38/STAT1/p21Waf1/Cip1 pathways and induce G1‐arrest and intrinsic apoptosis in breast carcinomas.

Worldwide, breast cancer (BCa) is the most common cancer in women. Among its subtypes, triple-negative breast cancer (TNBC) is an aggressive form associated with diminished survival. TNBCs are characterized by their absence, or minimal expression, of the estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (i.e. ER-/-, PR-/-, Her2-/Low). Consequently, treatment for this subtype of BCa remains problematic. Silibinin, a derivative of the flavonoid silymarin, is reported to have anticancer activities against hepatic and non-small cell lung cancers. We hypothesized that silibinin might inhibit cell-extracellular matrix interactions via the regulation, expression, and activation of STAT3 in TNBCs, which could directly inhibit metastasis in silibinin-treated BCa cells. Using proliferation assays, we found that exposure to silibinin at a concentration of 200 µM inhibited the proliferation of breast cancer (BCa) cells; this concentration also inhibited phosphorylation of STAT3 and its principal upstream kinase, Jak2. Furthermore, we found that silibinin inhibited the nuclear translocation of STAT3, as well as its binding to the MMP2 gene promoter. The ability of silibinin to inhibit metastasis was further studied using an in vitro invasion assay. The results confirm the role of STAT3 as a critical mediator in the invasive potential of BCa cells, and STAT3 knock-down resulted in inhibition of invasion. The invasion ability of silibinin-treated BCa cells was studied in detail with the expression of MMP2. Prevention of STAT3 activation also resulted in the inhibition of MMP2 expression. Use of a small interfering RNA to knock down STAT3 (siSTAT3) allowed us to confirm the role of STAT3 in regulating MMP2 expression, as well as the mechanism of action of silibinin in inhibiting MMP2. Taken together, we found that silibinin inhibits the Jak2/STAT3/MMP2 signaling pathway, and inhibits the proliferation, migration, and invasion of triple-negative BCa cells.

Sorghum is a principal cereal food in a number of parts of the world and is critical in folk medicine in Asia and Africa. However, its effects on bone are unknown. Growth hormone (GH) is a regulator of bone growth and bone metabolism. GH activates several signaling pathways, including the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathways, thereby regulating expression of genes, including insulin‑like growth factor (IGF)‑1. Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage, increasing the pool of IGF‑1 target cells, the mature osteoblasts. In the present study, the effects of Hwanggeumchal sorghum extracts (HSE) on GH signaling via the Jak/STAT pathway in osteoblasts were investigated. HSE was not observed to be toxic to osteoblastic cells and increased the expression of BMP7 and GH‑related proteins, including STAT5B, p‑STAT5B, IGF‑1 receptor (IGF-1R), growth receptor hormone (GHR) and Jak2 in MC3T3‑E1 cells. In addition, HSE increased BMP7 and GHR mRNA expression in MC3T3‑E1 cells. The expression of HSE‑induced BMP7 and GHR was inhibited by AG490, a Jak2 kinase inhibitor. The observations indicate that HSE‑induced signaling is similar to GH signaling via the GHR‑Jak2 signaling axis. Using small interference RNA (siRNA) analysis, STAT5B was found to play an essential role in HSE‑induced BMP7 and GH signaling in MC3T3‑E1 cells. Results of the current study indicate that HSE promotes bone growth through activation of STAT5B.

Osteoclast differentiation is dependent on the activities of receptor activator NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Given that RANKL plays a critical role in osteoclast formation and bone resorption, any new compounds found to alter its activity would be predicted to have therapeutic potential for disorders associated with bone loss. Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-documented anti-oxidant and anti-inflammatory properties; currently its effects on osteoclast differentiation are unknown. We sought to investigate whether MSM could regulate osteoclastogenesis, and if so, its mechanism of action. In this study, we investigated the effects of MSM on RANKL-induced osteoclast differentiation, together with STAT3's involvement in the expression of osteoclastic gene markers. These experiments were conducted using bone marrow derived macrophages (BMMs) and cell line material, together with analyses that interrogated both protein and mRNA levels, as well as signaling pathway activity. Although MSM was not toxic to osteoclast precursors, MSM markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclast formation, and bone resorptive activity. Additionally, the expression of several osteoclastogenesis-related marker genes, including TRAF6, c-Fos, NFATc1, cathepsin K, and OSCAR were suppressed by MSM. MSM mediated suppression of RANKL-induced osteoclastogenesis involved inhibition of ITAM signaling effectors such as PLCγ and Syk, with a blockade of NF-kB rather than MAPK activity. Furthermore, MSM inhibited RANKL-induced phosphorylation of STAT3 Ser727. Knockdown of STAT3 using shRNAs resulted in reduced RANKL-mediated phosphorylation of Ser727 STAT3, and TRAF6 in cells for which depletion of STAT3 was confirmed. Additionally, the expression of RANKL-induced osteoclastogenic marker genes were significantly decreased by MSM and STAT3 knockdown. Taken together, these results indicate that STAT3 plays a pivotal role in RANKL-induced osteoclast formation, and that MSM can attenuate RANKL-induced osteoclastogenesis by blocking both NF-kB and STAT3 activity.

Background Combination therapy, which reduces the dosage intensity of the individual drugs while increasing their efficacy, is not a novel approach for the treatment of cancer. Methylsulfonylmethane (MSM) is an organic sulfur compound shown to act against tumor cells. Tamoxifen is a commercially available therapeutic agent for breast malignancies. Methods In the current study, we analyzed the combinatorial effect of MSM and tamoxifen on the suppression of ER-positive breast cancer xenograft growth and metastasis. Additionally, we also validated the molecular targets by which the drug combination regulated tumor growth and metastasis. Results We observed that the combination of MSM and tamoxifen regulated cell viability and migration in vitro. The intragastric administration of MSM and subcutaneous implantation of tamoxifen tablets led to tumor growth suppression and inhibition of the Janus kinase 2 (Jak2)/signal transducer and activator of transcription 5b (STAT5b) pathway. Our study also assessed the regulation of signaling molecules implicated in the growth, progression, differentiation, and migration of cancer cells, such as Jak2, STAT5b, insulin-like growth factor-1Rβ, and their phosphorylation status. Conclusions Study results indicated that this combination therapy inhibited tumor growth and metastasis. Therefore, this drug combination may have a synergistic and powerful anticancer effect against breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1445-0) contains supplementary material, which is available to authorized users.

Human urinary bladder cancer is the fifth most common cancer, with a worldwide estimate of about two million patients. Recurrence after complete transurethral prostatic resection is the most important problem in therapy. Combination therapy is a new approach in the treatment of cancers that do not respond to current therapies. These therapies have many advantages over conventional therapies, such as fewer side-effects and greater efficiency. Research efforts using natural compounds for the elimination or growth suppression of the cancer arise from studies on methylsulfonylmethane (MSM). MSM is a natural sulfur compound with no side-effects. AG490 is a tyrosine kinase inhibitor that has been extensively used for inhibiting Jak2 in vitro and in vivo. In our study, the combinatorial effect of these two agents on human bladder cancer cell lines and xenografts was analyzed. We observed that the combination of AG490 and MSM inhibited cancer cell viability and cell migration in vitro. This combination inhibited VEGF mRNA expression in bladder cancer cell lines. In vivo experiments showed that oral administration of AG490 and MSM combination significantly inhibited the growth of tumor xenografts in mice. Our study clearly demonstrates that the predominant effect of this combination is the reduction of signaling molecules including STAT3, STAT5b, IGF-1R, VEGF and VEGF-R2 which are involved in the growth, progression and metastasis of human bladder cancer. The anti-metastatic ability of this drug combination is confirmed using metastatic animal models. Therefore, this combination could have the effect of genesistasis and powerful anticancer effects against bladder cancer.

Osteoblast differentiation is a critical step in the maintenance of bone homeostasis. Osteoblast differentiation is generally maintained by growth hormone (GH) and various other endocrine and autocrine/paracrine factors. JAK2-STAT5B pathway is a central axis in the mechanism of GH signaling. Similarly, the autocrine/paracrine signaling factor IGF-1 also mediates its effects through this pathway. Analysis on JAK2-STAT5B pathway showed its importance in the IGF-1/IGF-1R mediated regulation of gene expression and osteoblast differentiation. Persistent activation of STAT5B and inhibition of STAT5B degradation showed increased osteoblastic differentiation and STAT5B/Runx-2 activities. Conditional gene silencing studies showed the importance of the JAK2-STAT5B pathway in stimulation of other transcription factors and expression of various differentiation markers.

Hsp90α is a molecular chaperone protein involved in the structural maturation of oncogenic signaling proteins. Hsp90 was recently identified as an anticancer target; various studies are ongoing to find ways for managing cancer through Hsp90α. However, this approach is limited by reported side-effects. Hypoxia is a hallmark of solid tumors, including those of breast cancer and the extent of tumor hypoxia is associated with resistance to treatment and poor prognosis. One of the major signaling pathways in cancer cells, the Jak2/STAT5b pathway, has been found to be closely correlated with hypoxia. The objective of this study was to investigate the role of Jak2/STAT5b in the regulation of Hsp90α expression so that Hsp90α targeting can be achieved indirectly by modulating the Jak2/STAT5b pathway. We examined the role of the Jak2/STAT5b pathway in the expression of Hsp90α under hypoxic conditions by immunoblotting, reporter gene assays, EMSA and RNA interference analysis. With the help of in vivo models, we also analyzed the expression of Hsp90α in different parts of solid tumor tissues. We found a close association between hypoxic stress and Hsp90α expression. We also determined that STAT5b regulates the expression of Hsp90α during hypoxic stimulation. Under hypoxic conditions the expression of Hsp90α and STAT5b were proportional. siRNA analysis and nucleotide analysis showed that the promoter of Hsp90α has a STAT5b binding domain. Our work confirmed that STAT5b is one of the transcription factors that regulate Hsp90α. We, therefore, concluded that under hypoxic conditions, the Jak2/STAT5b pathway regulates Hsp90α expression and it could serve as a promising target for the treatment of solid tumors.

Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-known anti-oxidant properties and anti-inflammatory activities. But, its effects on bone are unknown. Growth hormone (GH) is regulator of bone growth and bone metabolism. GH activates several signaling pathways such as the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) pathway, thereby regulating expression of genes including insulin-like growth factor (IGF)-1. GH exerts effects both directly and via IGF-1, which signals by activating the IGF-1 receptor (IGF-1R). In this study, we investigated the effects of MSM on the GH signaling via the Jak/STAT pathway in osteoblasts and the differentiation of primary bone marrow mesenchymal stem cells (MSCs). MSM was not toxic to osteoblastic cells and MSCs. MSM increased the expression of GH-related proteins including IGF-1R, p-IGF-1R, STAT5b, p-STAT5b, and Jak2 in osteoblastic cells and MSCs. MSM increased IGF1R and GHR mRNA expression in osteoblastic cells. The expression of MSM-induced IGF-1R and GHR was inhibited by AG490, a Jak2 kinase inhibitor. MSM induced binding of STAT5 to the IGF-1R and increased IGF-1 and IGF-1R promoter activities. Analysis of cell extracts by immunoprecipitation and Western blot showed that MSM enhanced GH-induced activation of Jak2/STAT5b. We found that MSM and GH, separately or in combination, activated GH signaling via the Jak2/ STAT5b pathway in UMR-106 cells. Using siRNA analysis, we found that STAT5b plays an essential role in GH signaling activation in C3H10T1/2 cells. Osteogenic marker genes (ALP, ON, OCN, BSP, OSX, and Runx2) were activated by MSM, and siRNA-mediated STAT5b knockdown inhibited MSM-induced expression of osteogenic markers. Furthermore, MSM increased ALP activity and the mineralization of MSCs. Taken together, these results indicated that MSM can promote osteogenic differentiation of MSCs through activation of STAT5b.

Background Cancer is one of the highly virulent diseases known to humankind with a high mortality rate. Breast cancer is the most common cancer in women worldwide. Sorghum is a principal cereal food in many parts of the world, and is critical in folk medicine of Asia and Africa. In the present study, we analyzed the effects of HSE in metastatic breast cancer. Methodology/Principal Findings Preliminary studies conducted on MDA-MB 231 and MCF-7 xenograft models showed tumor growth suppression by HSE. Western blotting studies conducted both in vivo and in vitro to check the effect of HSE in Jak/STAT pathways. Anti-metastatic effects of HSE were confirmed using both MDA-MB 231 and MCF-7 metastatic animal models. These studies showed that HSE can modulate Jak/STAT pathways, and it hindered the STAT5b/IGF-1R and STAT3/VEGF pathways not only by down-regulating the expression of these signal molecules and but also by preventing their phosphorylation. The expression of angiogenic factors like VEGF, VEGF-R2 and cell cycle regulators like cyclin D, cyclin E, and pRb were found down-regulated by HSE. In addition, it also targets Brk, p53, and HIF-1α for anti-cancer effects. HSE induced G1 phase arrest and migration inhibition in MDA-MB 231 cells. The metastasis of breast cancer to the lungs also found blocked by HSE in the metastatic animal model. Conclusions/Significance Usage of HS as a dietary supplement is an inexpensive natural cancer therapy, without any side effects. We strongly recommend the use of HS as an edible therapeutic agent as it possesses tumor suppression, migration inhibition, and anti-metastatic effects on breast cancer

Breast cancer is the most aggressive form of all cancers, with high incidence and mortality rates. The purpose of the present study was to investigate the molecular mechanism by which methylsulfonylmethane (MSM) inhibits breast cancer growth in mice xenografts. MSM is an organic sulfur-containing natural compound without any toxicity. In this study, we demonstrated that MSM substantially decreased the viability of human breast cancer cells in a dose-dependent manner. MSM also suppressed the phosphorylation of STAT3, STAT5b, expression of IGF-1R, HIF-1 alpha, VEGF, BrK and p-IGF-1R and inhibited triple-negative receptor expression in receptor-positive cell lines. Moreover, expression in receptor-positive cell-lines. Moreover, MSM decreased the DNA-binding activities of STAT5b and STAT3, to the target gene promoters in MDA-MB 231 or co-transfected COS-7 cells. We confirmed that MSM significantly decreased the relative luciferase activities indicating crosstalk between STAT5b/IGF-1R, STAT5b/HSP90 alpha, and STAT3/VEGF. To confirm these findings in vivo, xenografts were established in Balb/c athymic nude mice with MDA-MB 231 cells and MSM was administered for 30 days. Concurring to our in vitro analysis, these xenografts showed decreased expression of STAT3, STAT5b, IGF-1R and VEGF. Through in vitro and in vivo analysis, we confirmed that MSM can effectively regulate multiple targets including STAT3/VEGF and STAT5b/IGF-1R. These are the major molecules involved in tumor development, progression, and metastasis: Thus, we strongly recommend the use of MSM as a trial drug for treating all types of breast cancers including triple-negative cancers.