Recombinant proteins are generally fused with solubility enhancer tags to improve the folding and solubility of the target protein of interest. However, the fusion protein strategy usually requires ...expensive proteases to perform in vitro proteolysis and additional chromatographic steps to obtain tag‐free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co‐expression of a site‐specific protease, simplify the recombinant protein purification process, and promote the screening of molecules that fail to remain soluble after tag removal. High yields of soluble target proteins have already been achieved using these protease co‐expression systems. Herein, we review approaches for controlled intracellular processing systems tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular processing of recombinant proteins regarding system design features, advantages, and limitations of the various strategies.
Recombinant protein production in different Escherichia coli strains is a cornerstone of modern biotechnology. Controlled intracellular processing (CIP) of recombinant passenger proteins that are fused to solubility enhancer proteins has been proposed as an alternative to obtaining satisfactory yields of soluble recombinant proteins without the need for significant post‐processing steps. In this manuscript, we review different genetic systems currently available for controlled intracellular processing of fusion recombinant proteins, regarding system design features, significant advantages, and limitations of the various strategies.
Autophagy is a physiological cellular process that is crucial for development and can occurs in response to nutrient deprivation or metabolic disorders. Interestingly, autophagy plays a dual role in ...cancer cells—while in some situations, it has a cytoprotective effect that causes chemotherapy resistance, in others, it has a cytotoxic effect in which some compounds induce autophagy-mediated cell death. In this review, we summarize strategies aimed at autophagy for the treatment of cancer, including studies of drugs that can modulate autophagy-mediated resistance, and/or drugs that cause autophagy-mediated cancer cell death. In addition, the role of autophagy in the biology of cancer stem cells has also been discussed.
Hepatic cancer is one of the main causes of cancer-related death worldwide. Cancer stem cells (CSCs) are a unique subset of cancer cells that promote tumour growth, maintenance, and therapeutic ...resistance, leading to recurrence. In the present work, the ability of a ruthenium complex containing 1,3-thiazolidine-2-thione (RCT), with the chemical formula Ru(tzdt)(bipy)(dppb)PF6, to inhibit hepatic CSCs was explored in human hepatocellular carcinoma HepG2 cells. RCT exhibited potent cytotoxicity to solid and haematological cancer cell lines and reduced the clonogenic potential, CD133+ and CD44high cell percentages and tumour spheroid growth of HepG2 cells. RCT also inhibited cell motility, as observed in the wound healing assay and transwell cell migration assay. RCT reduced the levels of Akt1, phospho-Akt (Ser473), phospho-Akt (Thr308), phospho-mTOR (Ser2448), and phospho-S6 (Ser235/Ser236) in HepG2 cells, indicating that interfering with Akt/mTOR signalling is a mechanism of action of RCT. The levels of active caspase-3 and cleaved PARP (Asp214) were increased in RCT-treated HepG2 cells, indicating the induction of apoptotic cell death. In addition, RCT modulated the autophagy markers LC3B and p62/SQSTM1 in HepG2 cells and increased mitophagy in a mt-Keima-transfected mouse embryonic fibroblast (MEF) cell model, and RCT-induced cytotoxicity was partially prevented by autophagy inhibitors. Furthermore, mutant Atg5-/- MEFs and PentaKO HeLa cells (human cervical adenocarcinoma with five autophagy receptor knockouts) were less sensitive to RCT cytotoxicity than their parental cell lines, indicating that RCT induces autophagy-mediated cell death. Taken together, these data indicate that RCT is a novel potential anti-liver cancer drug with a suppressive effect on CSCs.
Display omitted
•RCT exhibits potent cytotoxicity to solid and haematological cancer cells.•RCT causes elimination of hepatic CSCs from HCC HepG2 cells.•RCT inhibits HCC HepG2 cell motility.•RCT suppresses Akt/mTOR signalling in HCC HepG2 cells.•RCT induces apoptotic and autophagic cell death in HCC HepG2 cells.
Acute myeloid leukemia (AML) is the most lethal form of leukemia. Standard anti-AML treatment remains almost unchanged for decades. Tingenone (TG) and 22-hydroxytingenone (22-HTG) are quinonemethide ...triterpenes found in the Amazonian plant Salacia impressifolia (Celastraceae), with cytotoxic properties in different histological types of cancer cells. In the present work, we investigated the anti-AML action mechanism of TG and 22-HTG in the AML HL-60 cell line. Both compounds exhibited potent cytotoxicity in a panel of cancer cell lines. Mechanistic studies found that TG and 22-HTG reduced cell growth and caused the externalization of phosphatidylserine, the fragmentation of internucleosomal DNA and the loss of mitochondrial transmembrane potential in HL-60 cells. In addition, pre-incubation with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, prevented TG- and 22-HTG-induced apoptosis, indicating cell death by apoptosis via a caspase-dependent pathway. The analysis of the RNA transcripts of several genes indicated the interruption of the cellular antioxidant system, including the downregulation of thioredoxin, as a target for TG and 22-HTG. The application of N-acetyl-cysteine, an antioxidant, completely prevented apoptosis induced by TG and 22-HTG, indicating activation of the apoptosis pathway mediated by oxidative stress. Moreover, TG and 22-HTG induced DNA double-strand break and phosphorylation of JNK2 (T183/Y185) and p38α (T180/Y182), and co-incubation with SP 600125 (JNK/SAPK inhibitor) and PD 169316 (p38 MAPK inhibitor) partially prevented apoptosis induced by TG and 22-HTG. Together, these data indicate that TG and 22-HTG are new candidate for anti-AML therapy targeting thioredoxin.
•TG and 22-HTG induce caspase-mediated apoptotic cell death.•TG and 22-HTG target oxidative stress through downregulation of thioredoxin.•TG and 22-HTG cause DNA damage.•TG and 22-HTG cause JNK/p38-mediated apoptosis.
Common strategies to improve recombinant protein production in Escherichia coli often involve the test and optimization of several different variables, when using traditional expression vectors that ...are commercially available. Now, modern synthetic biology-based strategies allow for extensive modifications of these traditional vectors, or even construction of entirely new modular vectors, so as to permit tunable production of the recombinant proteins of interest. Herein, we describe the engineering of a new expression operating unit (EOU; 938 bp) for producing recombinant proteins in E. coli, through the combinatorial assembly of standardized and well-characterized genetic elements required for transcription and translation (promoter, operator site, RBS, junction RBS-CDS, cloning module, transcriptional terminator). We also constructed a novel T7 promoter variant with increased transcriptional activity (1.7-fold higher), when compared to the canonical wild type T7 promoter sequence. This new EOU yielded an improved production of the reporter protein superfolder GFP (sfGFP) in E. coli BL21(DE3) (relative fluorescence units/RFU = 70.62 ± 1.62 A U.) when compared to a high-producing control expression vector (plasmid BBa_I746909; RFU = 59.68 ± 1.82 A U.). The yields of purified soluble recombinant sfGFP were also higher when using the new EOU (188 mg L−1 culture vs. 108 mg L−1 in the control) and it performed similarly well when inserted into different plasmid backbones (pOPT1.0/AmpR and pOPT2.0/CmR).
•A new expression operating unit (EOU) for recombinant protein production in E. coli.•Combinatorial assembly of well-characterized transcription/translation elements.•Engineering of a novel T7 promoter variant with increased transcriptional activity.•Optimized production and purification of soluble recombinant superfolder GFP.
Herein, ruthenium complexes containing heterocyclic thioamidates Ru(mmi)(bipy)(dppb)PF
(
), Ru(tzdt)(bipy)(dppb)PF
(
), Ru(dmp)(bipy)(dppb)PF
(
) and Ru(mpca)(bipy)(dppb)PF
(
) were investigated for ...their cellular and molecular effects in cancer cell lines. Complexes
and
were the most potent of the four compounds against a panel of different cancer cell lines in monolayer cultures and showed potent cytotoxicity in a 3D model of multicellular spheroids that formed from human hepatocellular carcinoma HepG2 cells. In addition, both complexes were able to bind to DNA in a calf thymus DNA model. Compared to the controls, a reduction in cell proliferation, phosphatidylserine externalization, internucleosomal DNA fragmentation, and the loss of the mitochondrial transmembrane potential were observed in HepG2 cells that were treated with these complexes. Additionally, coincubation with a pan-caspase inhibitor (Z-VAD(OMe)-FMK) reduced the levels of apoptosis that were induced by these compounds compared to those in the negative controls, indicating that cell death through apoptosis occurred via a caspase-dependent pathway. Moreover, these complexes also induced the phosphorylation of ERK1/2, and coincubation with an MEK inhibitor (U0126), which is known to inhibit the activation of ERK1/2, but not JNK/SAPK and p38 MAPK inhibitors, reduced the complexes-induced apoptosis compared to that in the negative controls, indicating that the induction of apoptotic cell death occurred through ERK1/2 signaling in HepG2 cells. On the other hand, no increase in oxidative stress was observed in HepG2 cells treated with the complexes, and the complexes-induced apoptosis was not reduced with coincubation with the antioxidant N-acetylcysteine or a p53 inhibitor compared to that in the negative controls, indicating that apoptosis occurred via oxidative stress- and p53-independent pathways. Finally, these complexes also reduced the growth of HepG2 cells that were engrafted in C.B-17 SCID mice compared to that in the negative controls. These results indicated that these complexes are novel anticancer drug candidates for liver cancer treatment.
Cancer stem cells (CSCs) are defined as a rare population of cancer cells related to tumor initiation and maintenance. These cells are primarily responsible for tumor growth, invasion, metastasis, ...recurrence, and resistance to chemotherapy. In this paper, we demonstrated the ability of Ru(II)-based complexes containing 2-thiouracil derivatives with the chemical formulas trans-Ru(2TU)(PPh3)2(bipy)PF6 (1) and trans-Ru(6m2TU)(PPh3)2(bipy)PF6 (2) (where 2TU = 2-thiouracil and 6m2TU = 6-methyl-2-thiouracil) to suppress liver CSCs by targeting NF-κB and Akt/mTOR signaling. Complexes 1 and 2 displayed potent cytotoxic effects on cancer cell lines and suppressed liver CSCs from HepG2 cells. Increased phosphatidylserine exposure, loss of mitochondrial transmembrane potential, increased PARP (Asp214) cleavage, DNA fragmentation, chromatin condensation and cytoplasmic shrinkage were detected in HepG2 cells treated with these complexes. Mechanistically, complexes 1 and 2 target NF-κB and Akt/mTOR signaling in HepG2 cells. Cell motility inhibition was also detected in HepG2 cells treated with these complexes. Complexes 1 and 2 also inhibited tumor progression in mice with HepG2 cell xenografts and exhibited tolerable systemic toxicity. Taken together, these results indicate that these complexes are new anti-HCC drug candidates that can suppress liver CSCs.
Duguetia gardneriana, popularly known in the Brazilian northeast as "jaquinha", is a species belonging to the family Annonaceae. The aim of this work was to assess the chemical composition and ...antitumor properties of the essential oil from the leaves of D. gardneriana in experimental models. The chemical composition of the essential oil was analyzed via gas chromatography-flame ionization detector and gas chromatography-mass spectrometry. In vitro cytotoxic activity was determined in cultured tumor cells, and in vivo antitumor activity was assessed in B16-F10-bearing mice. The identified compounds were β-bisabolene (80.99%), elemicin (8.04%), germacrene D (4.15%), and cyperene (2.82%). The essential oil exhibited a cytotoxic effect, with IC50 values of 16.89, 19.16, 13.08, and 19.33 µg/mL being obtained for B16-F10, HepG2, HL-60, and K562 cell lines, respectively. On the other hand, β-bisabolene was inactive in all of the tested tumor cell lines (showing IC50 values greater than 25 µg/mL). The in vivo analysis revealed tumor growth inhibition rates of 5.37-37.52% at doses of 40 and 80 mg/kg/day, respectively. Herein, the essential oil from the leaves of D. gardneriana presented β-bisabolene as the major constituent and showed cytotoxic and antitumor potential.
Ruthenium-based compounds have gained great interest due to their potent cytotoxicity in cancer cells; however, much of their potential applications remain unexplored. In this paper, we report the ...synthesis of a novel ruthenium complex with xanthoxylin (RCX) and the investigation of its cellular and molecular action in human hepatocellular carcinoma HepG2 cells. We found that RCX exhibited a potent cytotoxic effect in a panel of cancer cell lines in monolayer cultures and in a 3D model of multicellular cancer spheroids formed from HepG2 cells. This compound is detected at a high concentration in the cell nuclei, induces DNA intercalation and inhibits DNA synthesis, arresting the cell cycle in the S-phase, which is followed by the activation of the caspase-mediated apoptosis pathway in HepG2 cells. Gene expression analysis revealed changes in the expression of genes related to cell cycle control, apoptosis and the MAPK pathway. In addition, RCX induced the phosphorylation of ERK1/2, and pretreatment with U-0126, an MEK inhibitor known to inhibit the activation of ERK1/2, prevented RCX-induced apoptosis. In contrast, pretreatment with a p53 inhibitor (cyclic pifithrin-α) did not prevent RCX-induced apoptosis, indicating the activation of a p53-independent apoptosis pathway. RCX also presented a potent in vivo antitumor effect in C.B-17 SCID mice engrafted with HepG2 cells. Altogether, these results indicate that RCX is a novel anticancer drug candidate.
Solubility tags are commonly fused to target recombinant proteins to enhance their solubility and stability. In general, these protein tags must be removed to avoid misfolding of the partner protein ...and to allow for downstream applications. Nevertheless,
tag removal increases process complexity and costs. Herein, we describe a synthetic biology-based strategy to permit
removal of a solubility tag (EDA, KDPG aldolase), through co-expression of the fusion recombinant protein (EDA-EGFP) and the tag-cleaving protease (TEVp), in a controlled manner. Basically, the system uses three repressor proteins (LacI, cI434, and TetR) to regulate the expressions of EDA-EGFP and TEVp, in a regulatory cascade that culminates with the release of free soluble target protein (EGFP), following a single chemical induction by IPTG. The system worked consistently when all biological parts were cloned in a single plasmid, pSolubility(SOL)A (7.08 Kb, Amp
), and transformed in
Rosetta (DE3) or BL21(DE3) strains. Total soluble recombinant protein yield (EDA-EGFP + free EGFP) was
. 272.0 ± 60.1 μg/mL of culture, following IMAC purification; free EGFP composed great part (average = 46.5%; maximum = 67.3%) of the total purified protein fraction and was easily separated from remaining fusion EDA-EGFP (53 KDa) through filtration using a 50 KDa cut-off centrifugal filter.
PDF
