Survivin Inhibition by Piperine Sensitizes Glioblastoma Cancer Stem Cells and Leads to Better Drug Response

Glioblastoma multiforme (GBM) cancer stem cells (GSCs) are one of the strongest contributing factors to treatment resistance in GBM. Identification of biomarkers capable of directly affecting these cells within the bulk tumor is a major challenge associated with the development of new targeting strategies. In this study, we focus on understanding the potential of the multifunctional extraordinaire survivin as a biomarker for GSCs. We analyzed the expression profiles of this gene using various publicly available datasets to understand its importance in stemness and other cancer processes. The findings from these studies were further validated using human GSCs isolated from a GBM cell line. In these GSCs, survivin was inhibited using the dietary phytochemical piperine (PIP) and the subsequent effects on stemness, cancer processes and Temozolomide were investigated. In silico analysis identified survivin to be one of the most significant differentially regulated gene in GSCs, in comparison to common stemness markers. Further validation studies on the isolated GSCs showed the importance of survivin in stemness, cancer progression and therapy resistance. Taken together, our study identifies survivin as a more consistent GSC marker and also suggests the possibility of using survivin inhibitors along with standard of care drugs for better therapeutic outcomes

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Not AvailableEpsilon toxin (Etx) belongs to family of pore-forming toxin and is produced by Clostridium perfringens type D. The Etx toxin is responsible for the pathogenesis of enterotoxaemia in sheep and goats, and occasionally in other livestock animals. The present study aimed to develop a Clostridium perfringens epsilon toxin-based chimeric epitope construct having immunodominant B-cell epitope and universal T-cell epitope and its immunogenicity was evaluated in mice and rabbit. An artificial chimeric epitope construct (CEC) was prepared by joining tandem repeats of a peptide containing amino acids (aa) 134-145 of epsilon toxin B-cell epitope and universal T-cell epitopes. The CEC was expressed in the Escherichia coli following codon optimization for efficient translational efficiency and purified by affinity chromatography. The antigenic reactivity of r-CEC proteins was confirmed by western blot with rabbit anti-r-Etox hyperimmune sera. The immunogenicity of the recombinant single CEC was examined in mice and rabbit by indirect ELISA. It was found that r-CEC yielded high titers of neutralizing antibodies (≥ 1.035 IU/ml) in immunized mice and rabbit. The potency of chimeric protein immunized serum was observed to be higher than the recommended level (0.1-0.3 IU/ml) for protection in sheep and goats. This indicated the potential ability of the chimeric protein as a vaccine candidate. This further requires studying the immune response in targeted host species (sheep and goat).Not Availabl

Objective assessment of endogenous collagen in vivo during tissue repair by laser induced fluorescence.

Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm2 He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications

Scoring parameters for the collagen assessment in histological sections.

<p>Scoring parameters for the collagen assessment in histological sections.</p

Qualitative scoring for the collagen assessment in histological sections of Un-wounded control.

<p>Note.</p><p>Size of the fiber: Th- Thick; Tn-Thin;</p><p>Dermal layers: P- Papillary; M- Mid; D- Deep;</p><p>Orientation of the collagen fiber: H- Horizontal; V- Vertical.</p

Qualitative scoring for the collagen assessment in histological sections of Un-illuminated control.

<p>Note.</p><p>Size of the fiber: Th- Thick; Tn-Thin;</p><p>Dermal layers: P- Papillary; M- Mid; D- Deep;</p><p>Orientation of the collagen fiber: H- Horizontal; V- Vertical.</p

Experimental design.

<p>Schematic layout of the experimental setup for recording <i>in vivo</i> autofluorescence and microscopic studies. Typical overlaid <i>ex vivo</i> and <i>in vivo</i> autofluorescence spectra of granulation tissue at 325 nm excitation with peaks for collagen (A) & NADH (B) shown in the inset.</p

Typical <i>in vivo</i> autofluorescence spectra.

<p>Typical normalized <i>in vivo</i> autofluorescence spectra of un-illuminated control, unwounded control and 2 J/cm² treated animals on day 0 (A), day 5 (B), day 10 (C), day 30 (D), day 45(E), and day 60 (F) post-wounding. Inset- Comparision of the spectral range 390–405 nm indicating the collagen peak of the all the experimental group animals.</p

Autofluorescence patterns with varying age and gender.

<p>Influence of age on autofluorescence pattern of collagen in un-wounded skin (A). Average collagen intensity was plotted against different age group (in weeks). Collagen intensity values with respect to normalized NADH levels for male and female animals of un-wounded control (B), un-illuminated control (C), and optimum laser dose treated group (D) on different post-wounding days.</p