Sarmistha Talukdar

Silk protein fibroin from Antheraea mylitta for cardiac tissue engineering

The human heart cannot regenerate after an injury. Lost cardiomyocytes are replaced by scar tissue resulting in reduced cardiac function causing high morbidity and mortality. One possible solution to this problem is cardiac tissue engineering. Here, we have investigated the suitability of non-mulberry silk protein fibroin from Indian tropical tasar Antheraea mylitta as a scaffold for engineering a cardiac patch in vitro. We have tested cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication and contractility of 3-days postnatal rat cardiomyocytes on silk fibroin. Our data demonstrate that A. mylitta silk fibroin exhibits similar properties as fibronectin, a component of the natural matrix for cardiomyocytes. Comparison to mulberry Bombyx mori silk protein fibroin shows that A. mylitta silk fibroin is superior probably due to its RGD domains. 3D scaffolds can efficiently be loaded with cardiomyocytes resulting in contractile patches. In conclusion, our findings demonstrate that A. mylitta silk fibroin 3D scaffolds are suitable for the engineering of cardiac patches.

Nonmulberry silk biopolymers

The silk produced by silkworms are biopolymers and can be classified into two types--mulberry and nonmulberry. Mulberry silk of silkworm Bombyx mori has been extensively explored and used for century old textiles and sutures. But for the last few decades it is being extensively exploited for biomedical applications. However, the transformation of nonmulberry silk from being a textile commodity to biomaterials is relatively new. Within a very short period of time, the combination of load bearing capability and tensile strength of nonmulberry silk has been equally envisioned for bone, cartilage, adipose, and other tissue regeneration. Adding to its advantage is its diverse morphology, including macro to nano architectures with controllable degradation and biocompatibility yields novel natural material systems in vitro. Its follow on applications involve sustained release of model compounds and anticancer drugs. Its 3D cancer models provide compatible microenvironment systems for better understanding of the cancer progression mechanism and screening of anticancer compounds. Diversely designed nonmulberry matrices thus provide an array of new cutting age technologies, which is unattainable with the current synthetic materials that lack biodegradability and biocompatibility. Scientific exploration of nonmulberry silk in tissue engineering, regenerative medicine, and biotechnological applications promises advancement of sericulture industries in India and China, largest nonmulberry silk producers of the world. This review discusses the prospective biomedical applications of nonmulberry silk proteins as natural biomaterials.

Engineered silk fibroin protein 3D matrices for in vitro tumor model.

3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.

Effect of initial cell seeding density on 3D-engineered silk fibroin scaffolds for articular cartilage tissue engineering

The repair of articular cartilage defects poses a continuing challenge. Cartilage tissue engineering through the culture of chondrocytes seeded in 3D porous scaffolds has the potential for generating constructs that repair successfully. It also provides a platform to study scaffold-cell and cell-cell interactions. The scaffold affects the growth and morphology of cells growing on it, and concomitantly, cells affect the properties of the resultant tissue construct. Silk fibroin protein from Antheraea mylitta, a non-mulberry Indian tropical tasar silkworm, is a potential biomaterial for diverse applications due to its widespread versatility as a mechanically robust, biocompatible, tissue engineering material. Analysis of silk fibroin scaffolds seeded with varying initial densities (25, 50 and 100 million cells/ml) and cultured for 2 weeks showed that thickness and wet weight increased by 60-70% for the highest cell density, and DNA, GAG and collagen content of the cartilaginous constructs increased with increasing cell density. Mechanical characterization of the constructs elucidated that the highest density constructs had compressive stiffness and modulus 4-5 times that of cell-free scaffolds. The present results indicate the importance of cell seeding density in the rapid formation of a functional cartilaginous tissue.

An Emerging Functional Natural Silk Biomaterial from the only Domesticated Non-mulberry Silkworm Samia ricini

Mulberry silk fibroin is a widely used biomaterial and recent work on non-mulberry silk fibroin also suggests it may have similar uses. We expect silk fibroin from the only domesticated non-mulberry eri silkworm, Samia ricini, to possess useful properties as a biomaterial. Eri silk gland fibroin is a heterodimeric protein of approximately 450 kDa. Cytocompatibility evaluation with fibroblasts and osteoblast-like cells shows good cell attachment, viability and proliferation. The matrices, which have high thermal stability and good swellability, are also haemocompatible. Eri silk production is cost effective as no agronomic practices are required for their host plant cultivation. This fibroin provide new opportunities as an alternative natural functional biomaterial in various biomedical applications.

Inhibition of radiation-induced glioblastoma invasion by genetic and pharmacological targeting of MDA-9/Syntenin

Significance In the setting of glioblastoma multiforme (GBM), invasion of cells into normal brain and the unlikeliness of complete surgical removal contributes to GBM lethality and recurrence. “Gold standard” GBM treatment includes adjuvant radiotherapy. Unfortunately, cells surviving radiation demonstrate increased invasion and therapeutic resistance. Melanoma differentiation-associated gene 9 (MDA-9/Syntenin) expression is elevated in patient-derived tumors and GBM cell lines, which correlates with decreased survival and poor response to radiation. Genetic suppression of MDA-9/Syntenin sensitizes GBM to radiation by inhibiting radiation-induced invasion gains and signaling changes. Additionally, intraperitoneal administration of a small-molecule MDA-9/Syntenin inhibitor, PDZ1i, developed using innovative fragment-based drug design and NMR approaches, improved survival of brain tumor-bearing mice. Survival was enhanced further when used with radiation, supporting MDA-9/Syntenin as a therapeutic target for this deadly disease. Glioblastoma multiforme (GBM) is an intractable tumor despite therapeutic advances, principally because of its invasive properties. Radiation is a staple in therapeutic regimens, although cells surviving radiation can become more aggressive and invasive. Subtraction hybridization identified melanoma differentiation-associated gene 9 [MDA-9/Syntenin; syndecan-binding protein (SDCBP)] as a differentially regulated gene associated with aggressive cancer phenotypes in melanoma. MDA-9/Syntenin, a highly conserved double-PDZ domain-containing scaffolding protein, is robustly expressed in human-derived GBM cell lines and patient samples, with expression increasing with tumor grade and correlating with shorter survival times and poorer response to radiotherapy. Knockdown of MDA-9/Syntenin sensitizes GBM cells to radiation, reducing postradiation invasion gains. Radiation induces Src and EGFRvIII signaling, which is abrogated through MDA-9/Syntenin down-regulation. A specific inhibitor of MDA-9/Syntenin activity, PDZ1i (113B7), identified through NMR-guided fragment-based drug design, inhibited MDA-9/Syntenin binding to EGFRvIII, which increased following radiation. Both genetic (shmda-9) and pharmacological (PDZ1i) targeting of MDA-9/Syntenin reduced invasion gains in GBM cells following radiation. Although not affecting normal astrocyte survival when combined with radiation, PDZ1i radiosensitized GBM cells. PDZ1i inhibited crucial GBM signaling involving FAK and mutant EGFR, EGFRvIII, and abrogated gains in secreted proteases, MMP-2 and MMP-9, following radiation. In an in vivo glioma model, PDZ1i resulted in smaller, less invasive tumors and enhanced survival. When combined with radiation, survival gains exceeded radiotherapy alone. MDA-9/Syntenin (SDCBP) provides a direct target for therapy of aggressive cancers such as GBM, and defined small-molecule inhibitors such as PDZ1i hold promise to advance targeted brain cancer therapy.

Silk gland fibroin from indian muga silkworm Antheraea assama as potential biomaterial

There is an increasing demand for new versatile biomaterials. Silk is reported by many researchers as an ideal biomaterial for different biomedical applications. Most of the studies are carried out on mulberry silk Bombyx mori, however silk from Indian non-mulberry silkworms are relatively unexplored. In this report we fabricate 2D matrices from the regenerated aqueous silk fibroin protein of the glands of non-mulberry Indian muga silkworm Antheraea assama (assamensis). Its biochemical, biophysical characteristics and its cytocompatibility for biomedical uses are evaluated. The properties of this muga gland fibroin are compared with silk gland fibroin from non-mulberry Indian tropical tasar silkworm Antheraea mylitta and with the fibroin from the cocoon of mulberry silkworm Bombyx mori. The gland fibroin of Antheraea assama is observed to consist of two polypeptides of approximately 250 kDa each linked by disulfide bond. Fourier transformed infrared spectroscopy and X-ray diffraction studies indicate random coil structure of dissolved fibroin solution. The alpha-helical structure in 2D films changed to beta-sheets upon ethanol treatment, imparting crystallinity and insolubility. The fibroin film is found to be the least hydrophilic, followed by B. mori and A. mylitta silk. Biocompatibility of the films from all three species is investigated through the cell attachment and spreading study of MG-63 human osteoblast-like cells. The cytocompatibility of non-mulberry fibroin matrices are comparable with that of standard tissue culture plates. The results indicate that the non-mulberry Indian muga silk gland fibroin is also suitable matrix as a natural biomaterial for tissue engineering applications.

mda-7/IL-24 Mediates Cancer Cell-Specific Death via Regulation of miR-221 and the Beclin-1 Axis.

Melanoma differentiation-associated gene-7/IL-24 (mda-7/IL-24) displays broad-spectrum anticancer activity in vitro, in vivo in preclinical animal models, and in a phase I/II clinical trial in patients with advanced cancers without harming normal cells or tissues. Here we demonstrate that mda-7/IL-24 regulates a specific subset of miRNAs, including cancer-associated miR-221. Either ectopic expression of mda-7/IL-24 or treatment with recombinant His-MDA-7 protein resulted in downregulation of miR-221 and upregulation of p27 and PUMA in a panel of cancer cells, culminating in cell death. Mda-7/IL-24-induced cancer cell death was dependent on reactive oxygen species induction and was rescued by overexpression of miR-221. Beclin-1 was identified as a new transcriptional target of miR-221, and mda-7/IL-24 regulated autophagy through a miR-221/beclin-1 feedback loop. In a human breast cancer xenograft model, miR-221-overexpressing MDA-MB-231 clones were more aggressive and resistant to mda-7/IL-24-mediated cell death than parental clones. This is the first demonstration that mda-7/IL-24 directly regulates miRNA expression in cancer cells and highlights the novelty of the mda-7/IL-24-miR-221-beclin-1 loop in mediating cancer cell-specific death. Cancer Res; 77(4); 949-59. ©2016 AACR.

Abrus agglutinin is a potent anti-proliferative and anti-angiogenic agent in human breast cancer.

Abrus agglutinin (AGG), a plant lectin isolated from the seeds of Abrus precatorius, has documented antitumor and immunostimulatory effects in murine models. To examine possible antitumor activity against breast cancer, we established human breast tumor xenografts in athymic nude mice and intraperitoneally administered AGG. AGG inhibited tumor growth and angiogenesis as confirmed by monitoring the expression of Ki‐67 and CD‐31, respectively. In addition, TUNEL positive cells increased in breast tumors treated with AGG suggesting that AGG mediates anti‐tumorigenic activity through induction of apoptosis and inhibition of angiogenesis. On a molecular level, AGG caused extrinsic apoptosis through ROS generation that was AKT‐dependent in breast cancer cells, without affecting primary mammary epithelial cells, suggesting potential cancer specificity of this natural compound. In addition, using HUVECs, AGG inhibited expression of the pro‐angiogenic factor IGFBP‐2 in an AKT‐dependent manner, reducing angiogenic phenotypes both in vitro and in vivo. Overall, the present results establish that AGG promotes both apoptosis and anti‐angiogenic activities in human breast tumor cells, which might be exploited for treatment of breast and other cancers.

Evolving Strategies for Therapeutically Targeting Cancer Stem Cells.

Cancer is a multifactor and multistep process that is affected intrinsically by the genetic and epigenetic makeup of tumor cells and extrinsically by the host microenvironment and immune system. A key component of cancer involves a unique subpopulation of highly malignant cancerous cells referred to as cancer stem cells (CSCs). CSCs are positioned at the apex of the tumor hierarchy with an ability to both self-renew and also generate non-CSC/differentiated progeny, which contribute to the majority of the tumor mass. CSCs undergo functional changes and show plasticity that is stimulated by specific microenvironmental cues and interactions in the tumor niche, which contribute to the complexity and heterogeneity of the CSC population. The prognostic value of CSCs in the clinic is evident since there are many examples in which CSCs serve as markers for poor patient prognosis. CSCs are innately resistant to many standard therapies and they display anoikis resistance, immune evasion, tumor dormancy, and field cancerization, which may result in metastasis and relapse. Many academic laboratories and biotechnology companies are currently focusing on strategies that target CSCs. Combination therapies, epigenetic modifiers, stemness inhibitors, CSC surface marker-based therapies, and immunotherapy-based CSC-targeting drugs are currently undergoing clinical trials. Potential new targets/strategies in CSC-targeted therapy include MDA-9/Syntenin (SDCBP), Patched (PTCH), epigenetic targets, noncoding RNAs, and differentiation induction. Defining ways of targeting and destroying CSCs holds potential to impact significantly on cancer therapy, including prevention of metastasis and cancer recurrence.