Caspases, a group of protease enzymes (cysteine proteases), exist as inactive zymogens in the cells and execute apoptosis (programmed cell death). Caspase‐3, an executioner caspase, plays an imperative role in apoptosis and becomes a primary target for cancer treatment. A number of analogues of quinazoline, quinazolinone, indoloquinazolines, quinone, naphthoquinones, pyrroloiminoquinones, styrylquinolines, tetheredtetrahydroquinoline, fluoroquinolone, thiosemicarbazones, benzotriazole, pyrimidines, chalcone, and carbazoles have been reported till date, representing caspase‐3 mediated apoptosis for cancer therapy. Simultaneously, plant isolates, including lysicamine, podophyllotoxin, and majoranolide, have also been claimed for caspase‐3‐mediated apoptosis‐induced cytotoxicity. Procaspase‐activating compound‐1 (PAC‐1) is the first FDA approved orphan drug, and its synthetic derivative WF‐208 also showed fascinating caspase‐3 mediated anticancer activity. Till date, a large number of compounds have been reported and patented for their caspase‐3‐mediated cytotoxicity and now scientist is also focusing to introduce new compounds in market to encompass anticancer activity.
Cell-derived
exosomes (30–200 nm) as biological “nanocarriers”
have attracted a great deal of interest for therapeutic applications
due to their ability to internalize in in vivo biological systems
(i.e., cells). Although they can be harvested from various sources
including stem cells, yet an appropriate isolation and characterization
protocol to obtain “pure” exosomal population is needed.
For potential clinical applications, understanding the functional
ability of exosomes and their purity, that is, free from microvesicles,
apoptotic bodies, and protein aggregates, is a pre-requisite. To achieve
high purity and yield of exosomes from human Wharton’s jelly-derived
mesenchymal stem cells (hWJ-MSCs) in the size range of 30–200
nm, we have performed and compared three isolation procedures: ultracentrifugation
(UC), sucrose cushion (SC), and commercially available reagent (CR).
The isolated exosomes were characterized using nanoparticle tracking
analysis (NTA), field emission scanning electron microscopy (FESEM),
and atomic force microscopy (AFM). Furthermore, to understand the
therapeutic potential of the hWJ-MSC-derived exosomes (hWJ-ME) to
target pancreatic tumor cells, the internalization efficacy has been
evaluated on the MiaPaCa-2 cell lines using confocal microscopy and
flow cytometry. The NTA results showed sucrose cushion to be an optimal
method for exosome isolation with high purity (86.8%), as compared
to UC (40.5%;
p
= 0.050) and CR (38%;
p
= 0.050). Optical analysis by FESEM and AFM revealed that SC-isolated
exosomes presented a spherical morphology, whereas UC- and CR-isolated
exosomes exhibited an uneven morphology. Furthermore, the data from
confocal images and flow cytometry showed that hWJ-ME were internalized
by MiaPaCa-2, demonstrating the feasibility of exosomes as a “potential
nanocarrier”. Thus, our study suggests that a combination of
NTA (yield), AFM (dimensions), and FESEM (morphology and topography)
could provide sensitive biophysical characterization of hWJ-ME. In
the future, enriched exosomes could be used as a delivery vehicle
to transport target-specific drugs or gene-silencing constructs to
tumors.
The present study was aimed to validate expression stability of 6 housekeeping genes (viz. YWHAZ, SDHA, GAPDH, RPS15, RPS18 and RN18S1) in the oocytes and embryos of different stages in buffalo. A modified Trizol protocol was optimized for RNA isolation from as few as five oocytes. The expression level of selected genes was studied by an optimized real time PCR using DCT method and their stability of expression was evaluated by Microsoft Excel based visual application, geNORM. The analysis revealed that the RPS15 and GAPDH were the most stable genes across different samples. Also, the geometric mean of three genes (i.e. RPS15,RPS18 and GAPDH) were found suitable for normalization of real time PCR data from buffalo oocytes⁄embryos. The information would help in more accurate interpretation of gene expression data from oocytes⁄embryos towards understanding the molecular events in these cells during development.
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