Specific Cytotoxic Effects of Parasporal Crystal Proteins Isolated from Native Saudi Arabian Bacillus thuringiensis Strains against Cervical Cancer Cells

Abstract

Currently, global efforts are being intensified towards the discovery of local Bacillus thuringiensis (Bt) isolates with unique anticancer properties. Parasporins (PS) are a group of Bt non-insecticidal crystal proteins with potential and specific in vitro anticancer activity. However, despite the significant therapeutic potential of PS-producing Bt strains, our current knowledge on the effects of these proteins is limited. Hence, the main objective of this study was to screen Bt-derived parasporal toxins for cytotoxic activities against colon (HT-29) and cervical (HeLa) cancerous cell lines. Nine non-larvicidal and non-hemolytic Bt strains, native to Saudi Arabia, were employed for the isolation of their parasporal toxins. 16S rDNA sequencing revealed a 99.5% similarity with a reference Bt strain. While PCR screening results indicated the absence of selected Cry (Cry4A, Cry4B, Cry10 and Cry11), Cyt (Cyt1 and Cyt2) and PS (PS2, PS3 and PS4) genes, it concluded presence of the PS1 gene. SDS-PAGE analysis revealed that proteolytically-cleavaged PS protein profiles exhibit patterns resembling those observed with PS1Aa1, with major bands at 56 kDa and 17 kDa (Bt7), and 41 kDa and 16 kDa (Bt5). Solubilized and trypsinized PS proteins from all Bt strains exhibited a marked and dose-dependent cytotoxicity against HeLa cancerous cells but not against HT-29 cells. IC50 values ranged from 3.2 (Bt1) to 14.2 (Bt6) with an average of 6.8 µg/mL. The observed cytotoxicity of PS proteins against HeLa cells was specific as it was not evident against normal uterus smooth muscle cells. RT-qPCR analysis revealed the overexpression of caspase 3 and caspase 9 by 3.7, and 4.2 folds, respectively, indicative of the engagement of intrinsic pathway of apoptosis. To the best of our knowledge, this is the first report exploring and exploiting the versatile repertoire of Saudi Arabian environmental niches for the isolation of native and possibly novel Saudi Bt strains with unique and specific anticancer activity. In conclusion, native Saudi Bt-derived PS proteins might have a potential to join the arsenal of natural anticancer drugs.

Glabralysins, Potential New β-Pore-Forming Toxin Family Members from the Schistosomiasis Vector Snail Biomphalaria glabrata

“…First, using recombinant proteins, the hemolytic activity of Glabralysins will be investigated, and then their cytotoxic/cytolytic effects on micro-organisms or pathogens will be analyzed, as was done for Biomphalysin [25]. Moreover, as some the members of the Bt β-PFT family (the parasporins) have selective against specific human tumor cell lines [90,[93][94][95][96][97], any possible anticancer activity of Glabralysins will be investigated, and any molecules showing a positive activity can be considered as potential new chemotherapeutic drugs [98][99][100].…”

Biochemical and Molecular Investigation of In Vitro Antioxidant and Anticancer Activity Spectrum of Crude Extracts of Willow Leaves Salix safsaf

“…Cell lines were cultured in DMEM/high glucose supplemented with 2 mM L-glutamine, 10% FBS and 1% penicillin/streptomycin. Then, sub-confluent cultures (80–90%) were trypsinized (Trypsin 0.05%/0.53 mM EDTA) and spilt depending on the seeding ratio recommended by ATCC [ 16 , 17 ].…”

“…Cytotoxic effects of crude willow fractions and extracts on viabilities of cancerous cells were investigated by assessing the ability of reducing enzymes present in viable cells to transform MTT into formazan crystals according to our previous reports [ 16 , 17 ]. Seven Salix safsaf fractions/extracts were initially screened for anti-proliferative activity at one concentration (50 μg/mL) against the above-mentioned cell lines (one-dose pre-screening).…”

“…Analyses of mRNA transcripts of key pro- and anti-apoptotic marker genes were conducted with MCF-7 cells exposed to 100, 400 or 800 µg/mL doses of F3 for 72 h [ 16 ]. Briefly, RNA was isolated by use of the Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the instructions of the manufacturer.…”

Synthesis and Bioactivity Assessment of Novel Spiro Pyrazole-Oxindole Congeners Exhibiting Potent and Selective in vitro Anticancer Effects

“…Briefly, cells (HepG2 and HCT) were seeded at a concentration of 1.2-1.8 × 10 3 cells/well in 6-well plates. After 48-h treatment, the collected cells were lysed and centrifuged at 10,000 rpm for 20 min at 4 • C. The protein concentration was measured in the supernatant by Bradford protein assay [34]. Volume containing 50 mg of total protein was incubated with 5 mL of caspase substrate in 100 mL of the reaction buffer at 37 • C for 1 h in the dark.…”