Glucose-methanol-based fed-batch fermentation for the production of recombinant human interferon gamma (rhIFN-γ) and evaluation of its antitumor potential.

Squamous cell carcinoma (SCC) is nonmelanoma skin cancer, which is very common in patients having T-cell immunosuppressant drugs. Anticancerous agents such as cytokines showed effective response on SCC. Human interferon-gamma (hIFN-γ), a type II cytokines, are having potent antiproliferative and immunomodulatory effects. In the current study, the fed-batch cultivation of recombinant Pichia pastoris was carried out, and its effect on cell biomass production, recombinant human interferon-gamma (rhIFN-γ) production, and the overflow metabolites was estimated. P. pastoris GS115 strain coexpressed with 6-phosphogluconolactonase (SOL3) and ribulose-phosphate 3-epimerase (RPE1) gene (GS115/rhIFN-γ/SR) resulted in 60 mg L-1 of rhIFN-γ production, which was twofold higher as compared with the production from GS115/rhIFN-γ strain. The antiproliferative potential of rhIFN-γ was examined on the human squamous carcinoma (A431) cell lines. Cells treated with 80 ng mL-1 of rhIFN-γ exhibited 50% growth inhibition by enhancing the production of intracellular reactive oxygen species levels and disrupting membrane integrity. Our findings highlight a state of art process development strategy for the high-level production of rhIFN-γ and its potential application as a therapeutic drug in SCC therapy.

The inhibitory effect of silk sericin against ultraviolet-induced melanogenesis and its potential use in cosmeceutics as an anti-hyperpigmentation compound.

Ultraviolet radiation (UVR)-induced redox imbalance in melanocytes triggers the activation of tyrosinase that results in melanogenesis and its related skin disorders. Supplementation of biological reductants or anti-tyrosinase compounds inhibits such melanogenesis. Silk sericin (SS), a globular protein, is known to possess antioxidant and anti-tyrosinase activities along with other biological attributes. However, its inhibitory activity against UVR-induced melanogenesis has yet to be explored. In the current study, we have scientifically explored the inhibitory activity of SS against UVR-induced melanogenesis. Anti-tyrosinase activity of SS was assessed using mushroom tyrosinase, showing that Antheraea assamensis sericin (AAS) and Philosamia ricini sericin (PRS) inhibited 50% of its activity. Inhibitory activity of SS against UVR-induced melanogenesis was assessed by measuring the cellular melanin content, intracellular tyrosinase activity, and reactive oxygen species (ROS) levels in mouse melanoma. SS pretreatment significantly reduced cellular melanin and ROS production in UV irradiated melanocytes compared with SS untreated cells. AAS treatment before UVA or UVB irradiation significantly inhibited tyrosinase activity. Rheological studies showed that the skin care formulation prepared by the addition of AAS to the basic formulation minimally affected its flow properties. Altogether, our results validate that AAS efficiently inhibited UVR-induced melanogenesis and it could be used as a potential antioxidant molecule in skin care cosmeceutics.

Inhibitory role of silk cocoon extract against elastase, hyaluronidase and UV radiation-induced matrix metalloproteinase expression in human dermal fibroblasts and keratinocytes.

Topical delivery of potent antioxidants maintain the redox balance of the skin, which leads to the downregulation of matrix metalloproteinase (MMP) expression and prevents UV radiation-induced photoaging. In this study, we aimed at investigating the inhibitory role of silk cocoon extract (SCE) isolated from the Antheraea assamensis (AA), Bombyx mori (BM), and Philosamia ricini (PR) silk varieties against UV radiation-induced MMP expression. Incubation of elastase and hyaluronidase with Antheraea assamensis silk cocoon extract (AASCE) caused 50% inhibition of activity. The assessment of total collagen content using the Sirius red assay showed that AASCE (10 µg mL-1) and Philosamia ricini silk cocoon extract (PRSCE at 100 µg mL-1 concentration) post-treatment significantly enhanced the total collagen content in UVA1 and UVB irradiated HDF cells, whereas BM silk cocoon extract (BMSCE at 100 µg mL-1 concentration) post-treatment significantly enhanced the total collagen content in UVA1-irradiated HDF cells. Gene expression studies revealed AASCE and PRSCE post-treatment downregulated the expression of interleukin (IL)-6, MMP-1 and upregulated procollagen genes in UV irradiated HDF cells. Gelatin zymography studies with AASCE post-treatment downregulated the release of MMP-2 and MMP-9 by HaCaT cells. The overall results validate AASCE efficiently shielding UV radiation-induced collagen and elastin degradation by downregulation of MMP expression, substantiating its further use as a potent antioxidant complement in skin care formulations.

Silk sericin induced pro-oxidative stress leads to apoptosis in human cancer cells.

Pro-oxidative stress induced by dietary polyphenols elevates reactive oxygen species (ROS) production in cancer cells, which subsequently leads to oxidative stress-mediated apoptosis. Sericin, a principal component of silk is associated with a mixture of polyphenols and flavonoids, possesses various biomedical attributes including anticancer activity. In the present study, we have evaluated the pro-oxidative effect of Bombyx mori sericin (BMS), Antheraea assamensis sericin (AAS), and Philosamia ricini sericin (PRS) against different cancer cells. Cytotoxicity of silk sericin (SS) evaluated using A431, SAS, and MCF-7 cells showed ≥50% reduction in their viability at 4 mg/mL. Intracellular ROS levels, cell cycle arrest, and apoptosis assessed using flow cytometry corroborated that SS treatment elevated the intracellular ROS levels, caused cell cycle arrest at the sub-G1 phase and resulted in apoptotic cell death. SS treated A431 and SAS cells showed upregulation of p53 and dysregulation of Bax and Bcl-2 gene expression. Whereas, AAS treated MCF-7 cells showed upregulation of Bax and downregulation of Bcl-2 gene expression. AAS treated MCF-7 and SAS cells showed downregulation of Bcl-2 protein expression in comparison to their control cells. Thus, the present study demonstrates that the pro-oxidative effect induced by SS suppresses the cancer growth indicating its potential anticancer activity.

Protective Activity of Silk Sericin against UV Radiation-Induced Skin Damage by Downregulating Oxidative Stress.

Topical delivery of potential antioxidants protects the skin against ultraviolet (UV) radiation-induced oxidative damage through maintaining redox balance. Sericin, one of the major components of silk, possesses antioxidant property along with skin-protective activity against UVB radiation-induced damage. However, the protective activity of silk sericin (SS) extracted from different sources has not been explored against UVA and UVB radiation-induced oxidative damage. In the present study, we have systematically investigated the protective activity of sericin against UVA and UVB radiation-induced skin damage. MTT and neutral red assays showed that Philosamia ricini sericin (PRS) and Antheraea assamensis sericin (AAS) (10 µg/mL) treatment prior to UVA (12 J/cm2) and UVB (120 mJ/cm2) irradiations enhanced the viability of human keratinocytes. Examination of cell cycle arrest and apoptotic/necrotic cell death using flow cytometry showed that sericin treatment before UVA and UVB irradiation protected the cells from apoptotic cell death by arresting the cell cycle at G1 phase. Sericin pretreatment downregulated the interleukin (IL)-6 and IL-8, upregulated p53 and decrease the dysregulation of Bcl-2/Bax gene expression. AAS treatment prior to UVB irradiation significantly reduced skin inflammation, DNA fragmentation, and lipid peroxidation in the female SKH-1 hairless mouse skin. Altogether, our results substantiate the use of AAS in effectively ameliorating UVA and UVB radiation-induced skin damage, which holds prospects as a potent antioxidant supplement in the preparation of skin care products.

A novel reverse micellar purification strategy for histidine tagged human interferon gamma (hIFN-γ) protein from Pichia pastoris.

In the present study, we have demonstrated the process development of human interferon gamma (hIFN-γ) (upstream to downstream). The codon optimized hIFN-γ gene was cloned in Pichia pastoris X-33 and the expression was evaluated in batch reactor study. The purification was carried out with modified nickel chelated reverse micellar system and compared with the existing Nickle- Nitrilotriacetic acid (NI-NTA) method. The parameter optimization for forward extraction demonstrated a significant enhancement of 72% in forward extraction efficiency (FEE). Furthermore, the factors governing back extraction efficiency (BEE) were also optimized with sequential optimization involving Taguchi orthogonal array and Artificial Neural Network linked Simulated Annealing Algorithm (ANN-SA). The optimization resulted in 91.2% back extraction efficiency of recombinant human interferon gamma (rhIFN-γ). The development of this purification system with optimized parameters led to an efficient recovery of 67.3% and improved purity of 79.54%. Alongside, the anti-proliferative activity in MCF-7 cell lines were also investigated and it demonstrated that at 60ngmL-1 concentration of rhIFN-γ more that 25%.

Antioxidant potential of mulberry and non-mulberry silk sericin and its implications in biomedicine.

Sericin, a principal constituent of silk, is widely used in various biomedical applications. In addition, conferring protection against free radicals and oxidative damage add more value to its therapeutic potential. However, the antioxidant (AO) properties of silk sericin (SS) remains contingent on extraction procedures. In the present study, we have evaluated the effect of different extraction methods (conventional, autoclaving, urea, alkali and acid-degradation) on AO properties of SS from three Indian silk varieties [Antheraea assamensis (AA), Philosamia ricini (PR) and Bombyx mori (BM)]. The physico-chemical characterization studies revealed that the molecular weight of SS isolates of each method ranged from 10 to 220kDa along with varied protein structural biochemistry. SS extracts using urea-degradation (BM, PR and AA), conventional method and alkali-degradation (BM) displayed high percentage of ß-sheets, random coils and turns. Acid-degraded SS (PR, followed by AA and BM) showed the highest total flavonoid content while conventional method (PR), autoclaving (AA) and alkali-degradation (BM) displayed lowest flavonoid levels. Interestingly, SS extracted by autoclaving (BM and AA), acid-degradation (PR), conventional and alkali-degradation (BM, AA and PR) methods exhibited 50% reduction of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical. Moreover, the efficacy of antioxidant potential of SS extracted by different methods was found to be in the order of "alkali>autoclaving>conventional" as demonstrated in L929 cells. Correspondingly, the anti-lipid peroxidation activity of SS extracted by alkali method (AA, BM and PR) further confirmed better AO properties amid others. Thus, the present study demonstrates that the extraction methods may significantly affect AO activity of SS which might be of importance for potential cosmetic applications.

Mimicking Form and Function of Native Small Diameter Vascular Conduits Using Mulberry and Non-mulberry Patterned Silk Films.

Autologous graft replacement as a strategy to treat diseased peripheral small diameter (≤6 mm) blood vessel is often challenged by prior vein harvesting. To address this issue, we fabricated native-tissue mimicking multilayered small diameter vascular graft (SDVG) using mulberry (Bombyx mori) and Indian endemic non-mulberry (Antheraea assama and Philosamia ricini) silk. Patterned silk films were fabricated on microgrooved PDMS mold, casted by soft lithography. The biodegradable patterned film templates with aligned cell sheets were rolled onto an inert mandrel to mimic vascular conduit. The hemocompatible and mechanically strong non-mulberry films with RGD motif supported ∼1.2 folds greater proliferation of vascular cells with aligned anchorage. Elicitation of minimal immune response on subcutaneous implantation of the films in mice was complemented by ∼45% lower TNF α secretion by in vitro macrophage culture post 7 days. Pattern-induced alignment favored the functional contractile phenotype of smooth muscle cells (SMCs), expressing the signature markers-calponin, α-smooth muscle actin (α-SMA), and smooth muscle myosin heavy chain (SM-MHC). Endothelial cells (ECs) exhibited a typical punctuated pattern of von Willebrand factor (vWF). Deposition of collagen and elastin by the SMCs substantiated the aptness of the graft with desired biomechanical attributes. Furthermore, the burst strength of the fabricated conduit was in the range of ∼915-1260 mmHg, a prerequisite to withstand physiological pressure. This novel fabrication approach may eliminate the need of maturation in a pulsatile bioreactor for obtaining functional cellular phenotype. This work is thereby an attestation to the immense prospects of exploring non-mulberry silk for bioengineering a multilayered vascular conduit similar to a native vessel in "form and function", befitting for in vivo transplantation.