Subhayan Das

Probing the potential of apigenin liposomes in enhancing bacterial membrane perturbation and integrity loss.

Along with discovery of new antibacterial agents, it is important to develop novel drug delivery systems to effectively deliver drugs within bacterial cells for enhanced therapeutic activity. Liposomes have been extensively investigated as pharmaceutical carriers for improvement of therapeutic index of antimicrobial agents. The aim of this present study was to evaluate the antibacterial activity of free and liposomal formulation of apigenin, a plant based isoflavone and elucidate the mode of action. Distearoylphosphatidylcholine liposomes were prepared having nano-range particle size (104.3±1.8 nm), narrow particle distribution (0.204) and high encapsulation efficiency of apigenin (89.9±2.31%). Antibacterial activity of apigenin and efficacy of liposome-mediated apigenin delivery were determined from minimum inhibitory concentration values. Interaction studies using electron microscopy revealed adherence and fusion of liposomal apigenin with the bacteria causing membrane perturbation through reactive oxygen species generation which was evaluated by epi-fluorescence microscopy and fluorescence activated cell sorting. The interaction of apigenin liposomes with bacterial membrane increased intracellular drug concentration and thus, can be employed to deliver apigenin within cells to augment its antibacterial activity. Increased efficacy and hemocompatibility of this formulation paves way for future evaluation of underlying molecular mechanisms and in vivo testing for enhanced therapeutic effects.

S100A7 has an oncogenic role in oral squamous cell carcinoma by activating p38/MAPK and RAB2A signaling pathway

Oral cancer consists of squamous cell carcinoma within the oral cavity or on the lip. The clinical prognosis of this cancer is mostly poor owing to delayed diagnosis and a lack of appropriate early detection biomarkers to identify the disease. In the current study, we investigated the role of the S100A7 calcium-binding protein in oral squamous cell carcinoma as an activator of the p38/MAPK and RAB2A signaling pathway. The aim of the present study was to determine whether S100A7 and RAB2A have a role in tumor progression and to assess their potential as early detection biomarkers for oral cancer. This study elucidated the functional and molecular mechanisms of S100A7 and RAB2A activity in oral cancer, leading us to conclude that S100A7 is the major contributing factor in the occurrence of oral cancer and promotes local tumor progression by activating the MAPK signaling pathway via the RAB2A pathway. We hypothesize that S100A7 affects cell motility and invasion by regulating the RAB2A-associated MAPK signaling cascades. Also, the downregulation of S100A7 expression by RNA interference-mediated silencing inhibits oral cancer cell growth, migration and invasion.

Cooperative effect of BI-69A11 and celecoxib enhances radiosensitization by modulating DNA damage repair in colon carcinoma

Amplification of PI3K-Akt pathway promotes radioresistance in various cancers including colorectal carcinoma. Local recurrence in colon cancer causes poor prognosis affecting overall survival of cancer-affected patient population. To avoid local recurrence, pre-operative or post-operative additional radiotherapy is given. However, main concern regarding radiotherapy is to increase the radiosensitivity of malignant cell without hampering the activities of normal cells. In this context, addition of two or more than two chemotherapeutic drugs as a radiosensitizer is a common practice in radiation biology. BI-69A11 earlier showed potential apoptosis-inducing effect in melanoma and colon carcinoma. Celecoxib showed anti-cancer effects in both COX-2 dependent and independent pathways and used to act as a radiosensitizing enhancer. Here, we suggest that the combination of BI-69A11 and celecoxib inhibits the phosphorylation of ataxia telangiectasia mutated (ATM) kinase and DNA-PK responsible for ionizing radiation (IR)-induced double-strand break (DSB) repair. Moreover, the combinatorial effect of BI-69A11 and celecoxib attenuates the IR-induced G2/M cell cycle arrest. Furthermore, this combination also impairs IR-induced activation of Akt and downstream targets of ATM. This might lead to induced activation of apoptotic pathway after triple therapy treatment modulating pro-apoptotic and anti-apoptotic proteins. This activation of apoptotic pathway also showed the interdependence of PUMA and BAD in triple combination-treated colon cancer cells in a p53 independent manner. This study reveals the therapeutic potential of the triple combination therapy in prevention of radioresistance. Besides, it also demonstrates the cytotoxic effects of triple combination therapy in colon cancer. This study shows utility and potential implication on safety of the patients undergoing radiation therapy.

Pre-clinical risk assessment and therapeutic potential of antitumor lipopeptide ‘Iturin A’ in an in vivo and in vitro model

Lipopeptides are versatile bio-active weapons having antifungal, antibacterial, antimycoplasma and anticancer properties. In this study, the therapeutic potential and safety assessment of a lipopeptide molecule ‘Iturin A’ were evaluated. Iturin A was found to inhibit in vivo tumor growth in a sarcoma 180 mouse xenograft model. The antitumor efficacy of Iturin A was correlated with increased DNA fragmentation and modulation of CD-31, Ki-67, P-Akt, P-MAPK, apoptotic and anti-apoptotic proteins. Further, safety assessment was carried out in Sprague Dawley rats by 28 days repeated dose (28 days) toxicity and a bio-distribution study. In the toxicity study, Iturin A (10, 20 and 50 mg per kg per day) was administered to the animals for 28 days. Another group was kept for another 14 days without drug exposure after 28 days of treatment to access the reversibility of the toxicity. At the end of the treatment, body weight, food and water intake, organ weight, motility, hematology, serum biochemistry and histopathology of the major organs were evaluated. The bio-distribution of Iturin A was also performed in plasma as well as in different major organs by a well-developed and validated administration of Iturin A radiolabeled with 99mTc. The in vitro cytotoxic effect of Iturin A was also evaluated in BRL-3A rat liver cells. In the treated groups, various toxicities were found in the liver and spleen. However, these adverse effects were transient and reversible after discontinuation of Iturin A treatment. In conclusion, this pre-clinical study offered a preliminary investigation regarding the efficacy and safety assessment of Iturin A.

Prospects of nonmulberry silk protein sericin-based nanofibrous matrices for wound healing – In vitro and in vivo investigations

Recently, the progress in biomaterials for biomedical applications brings the focus of the research community toward nanomaterials. The nanofibrous matrices offer certain advantages (structural similarity to extracellular matrix, high surface area-to-volume ratio, increased elasticity, biostability, and strength) compared to other prevalent type of materials. This affirms their superiority and flexibility to be used in regenerative medicine. We have fabricated nonmulberry (Antheraea mylitta) silk protein sericin-based nanofibrous matrices (fiber thickness; 80-400 nm) with improved mechanical strength and desired stability (>4 weeks) as required for tissue reconstruction. These matrices support the adhesion, proliferation, and cellular interconnection of human keratinocytes. These are minimally hemolytic, nonimmunogenic, and capable of wound healing in vivo. Antibiotic (cephalexin hydrate [CH])-loaded nanofibrous matrices accelerate the full-thickness wound repair with minimal inflammation and without any signs of infection. The histological analysis authenticates skin restoration with re-epithelialization, generation of associated skin appendages, and synthesis of dense collagen fibrils. In addition, analysis of inflammatory genes and immunohistochemical assays have proved their biocompatibility and wound healing potential. Angiogenesis is also prevalent in the animal tissue treated with nanofibrous matrices. The results of in vitro and in vivo experimentations indicate a clear prospect of the fabricated sericin-based nanofibrous matrices to be used for skin regeneration. STATEMENT OF SIGNIFICANCE Nonmulberry silk protein sericin-based nanofibrous matrix is a useful biomaterial for wound healing, collagen production, and skin tissue repair. It has been used in different formulations including hydrogels and nanofibrous membranes with chitosan (CS) and polyvinyl alcohol (PVA). No experiments have been carried out to evaluate sericin-based nanofibrous membranes for skin tissue engineering application. The present study shows that the nanofibrous matrices fabricated by electrospinning nonmulberry silk protein sericin with CS and PVA mimic the architectural environment of the extracellular matrix fibrils. These matrices are minimally hemolytic, are nonimmunogenic, and support better growth of human keratinocytes in vitro and wound healing in vivo with re-epithelialization of the skin tissue and angiogenesis. This work indicates that these nonmulberry sericin-based nanofibrous matrices with CS may be used as an ideal physical environment and biological cues for the promotion of skin tissue reconstruction and repair.

Bioimpedimetric analysis in conjunction with growth dynamics to differentiate aggressiveness of cancer cells

Determination of cancer aggressiveness is mainly assessed in tissues by looking at the grade of cancer. There is a lack of specific method to determine aggressiveness of cancer cells in vitro. In our present work, we have proposed a bio-impedance based non-invasive method to differentiate aggressive property of two breast cancer cell lines. Real-time impedance analysis of MCF-7 (less aggressive) and MDA-MB-231 cells (more aggressive) demonstrated unique growth pattern. Detailed slope-analysis of impedance curves at different growth phases showed that MDA-MB-231 had higher proliferation rate and intrinsic resistance to cell death, when allowed to grow in nutrient and space limiting conditions. This intrinsic nature of death resistance of MDA-MB-231 was due to modulation and elongation of filopodia, which was also observed during scanning electron microscopy. Results were also similar when validated by cell cycle analysis. Additionally, wavelet based analysis was used to demonstrate that MCF-7 had lesser micromotion based cellular activity, when compared with MDA-MB-231. Combined together, we hypothesize that analysis of growth rate, death resistance and cellular energy, through bioimpedance based analysis can be used to determine and compare aggressiveness of multiple cancer cell lines. This further opens avenues for extrapolation of present work to human tumor tissue samples.

Multi-nucleated cells use ROS to induce breast cancer chemo-resistance in vitro and in vivo

Although there is a strong correlation between multinucleated cells (MNCs) and cancer chemo-resistance in variety of cancers, our understanding of how multinucleated cells modulate the tumor micro-environment is limited. We captured multinucleated cells from triple-negative chemo-resistant breast cancers cells in a time frame, where they do not proliferate but rather significantly regulate their micro-environment. We show that oxidatively stressed MNCs induce chemo-resistance in vitro and in vivo by secreting VEGF and MIF. These factors act through the RAS/MAPK pathway to induce chemo-resistance by upregulating anti-apoptotic proteins. In MNCs, elevated reactive oxygen species (ROS) stabilizes HIF-1α contributing to increase production of VEGF and MIF. Together the data indicate, that the ROS-HIF-1α signaling axis is very crucial in regulation of chemo-resistance by MNCs. Targeting ROS-HIF-1α in future may help to abrogate drug resistance in breast cancer.

Abstract 4105: Diosgenin and temozolamide: A potential combinatorial chemotherapy to overcome temozolamide resistance in glioblastoma multiforme

Introduction: Glioblastoma multiforme (GBM) accounts for the most aggressive form of tumor showing poor prognosis. Prevailing treatment modality includes chemotherapy with temozolomide (TMZ) concomitant with surgical resection and/or irradiation. However, it has been observed that a number of patients are developing resistance to TMZ owing to its high dosage regimen. The aim of this study is to examine the effects of diosgenin (DSG), a natural steroidal saponin obtained from fenugreek, in combination with TMZ in human GBM cells and TMZ resistant GBM cells. Methodology: The potential of combinatorial chemotherapy for overcoming TMZ resistance was evaluated through development of TMZ resistant GBM clones. These clones were generated by treatment of GBM cells with sub-lethal dose of TMZ over several cycles. Cellular effects were studied by viability assay, flow cytometry and wound healing assay on both single and combined drug treated GBM cells and TMZ resistant cells, respectively. The morphological study and cellular uptake in combined or individual drug treated cells was assessed by microscopy and immunofluorescence staining. The time dependant effect of drugs on invasive and migratory potential of GBM cells and TMZ resistant cells was analysed through zymography for matrix metalloproteinases (MMPs) activity and western blot for apoptotic proteins, epithelial and mesenchymal (EMT) markers, MMPs and VEGF expression. Further, the drug induced apoptosis was also assessed through chromatin condensation and DNA laddering assay. Results: Our investigation shows that co-administration of DSG and TMZ resulted in a substantial increase in GBM cell apoptosis and marked inhibition of cell growth in vitro. Anti-angiogenic and anti-invasive potential of DSG and TMZ were assessed through in vitro studies. At molecular level, DSG and TMZ synergistically lower XIAP expression and cleavage of intracellular death substrates such as PARP thereby shifting the balance from survival to apoptosis as indicated by the rise in the sub-G1 cell population. This combination also alters EMT markers, downregulates the expression of Bcl-xL, Bcl-2, Mcl1, MMPs and VEGF and induces expression of Bax, AIF, cytochrome C. Conclusion: The results suggested that DSG in combination with TMZ reduced the dose of TMZ, when administered singly, and also inhibited the migration and invasion of GBM and TMZ resistant cells. It induced apoptosis and altered the expression of EMT markers. These findings reveal a new therapeutic potential for overcoming TMZ resistance in GBM therapy. This novel modality may be a promising tool for GBM treatment. Citation Format: Y Rajesh, Angana Biswas, Subhayan Das, Mahitosh Mandal. Diosgenin and temozolamide: A potential combinatorial chemotherapy to overcome temozolamide resistance in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2017-4105

A self-assembled clavanin A-coated amniotic membrane scaffold for the prevention of biofilm formation by ocular surface fungal pathogens

Abstract Amniotic membrane (AM) is frequently used in ophthalmologic surgery for rapid ocular surface reconstruction. Sometimes it may create a major problem with associated infections after biofilm formation over the membrane. To overcome this problem, AM was coated with the antimicrobial peptide clavanin A. The antifungal activity of clavanin A in the native and self-assembled form was determined against the common ocular surface pathogens Candida albicans, Aspergillus fumigatus, Alternaria sp. and Fusarium sp. Biofilm formation over the coated surface was significantly reduced in comparison with the uncoated membrane. The coated membrane revealed effectiveness in terms of biocompatibility, cell attachment colonization when tested in non-cancerous 3T3 and human embryonic kidney (HEK)-293 cell lines. Clavanin A-coated AM also exhibited excellent physical, morphological and antifungal characteristics, indicating potential applicability for ocular surface infection control.