ABSTRACT
Arginase is a therapeutic enzyme for arginine-auxotrophic cancers but their low anticancer activity, less proteolytic tolerance and shorter serum half-life are the major shortcomings. In this study, arginase from Pseudomonas aeruginosa IH2 was purified to homogeneity and estimated as 75 kDa on native-PAGE and 37 kDa on SDS-PAGE. Arginase showed optimum activity at pH 8 and temperature 35 °C. Mn2+ and Mg2+ ions enhanced arginase activity while, Li+, Cu2+, and Al3+ ions reduced arginase activity. In-vitro serum half-life of arginase was 36 h and proteolytic half-life against trypsin and proteinase-K was 25 and 29 min, respectively. Anticancer activity of arginase was evaluated against colon, breast, leukemia, and prostate cancer cell lines and lowest IC50 (0.8 IU ml-1) was found against leukemia cell line HL-60. Microscopic studies and flow cytometric analysis of Annexin V/PI staining of HL-60 cells revealed that arginase induced apoptosis in dose-dependent manner. Cell cycle analysis suggested that arginase induced cell cycle arrest in G0/G1 phase. The increasing level of MMP loss, ROS generation and decreasing level of SOD, CAT, GPx and GSH suggested that arginase treatment triggered dysfunctioning of mitochondria. The cleavage of caspase-3, PARP-1, activations of caspase-8, 9 and high expression of proapoptotic protein Bax, low expression of anti-apoptotic protein Bcl-2 indicated that arginase treatment activates mitochondrial pathway of apoptosis. Purified arginase did not exert cytotoxic effects on human noncancer cells. Our study strongly supports that arginase could be used as potent anticancer agent but further studies are required which are underway in our lab.
Subject(s)
Apoptosis/drug effects , Arginase/toxicity , Bacterial Proteins/toxicity , G1 Phase Cell Cycle Checkpoints/drug effects , Membrane Potential, Mitochondrial/drug effects , Pseudomonas aeruginosa/enzymology , Arginase/chemistry , Arginase/isolation & purification , Arginase/pharmacokinetics , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/pharmacokinetics , Caspases/metabolism , Catalase/metabolism , Cell Line, Tumor , DNA Damage/drug effects , Down-Regulation/drug effects , HL-60 Cells , Humans , Hydrogen-Ion Concentration , Microscopy, Phase-Contrast , Molecular Weight , Protein Stability , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Substrate Specificity , Superoxide Dismutase/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
The serine/threonine protein kinase B (PKB), also known as Akt, is one of the multifaceted kinases in the human kinome, existing in three isoforms. PKB plays a vital role in phosphoinositide 3-kinase (PI3K)-mediated oncogenesis in various malignancies and is one of the attractive targets for cancer drug discovery. Recent studies have shown that the functional significance of an individual isoform of PKB is not redundant in cancer. It has been found that PKB isoforms play distinct roles in the regulation of cellular invasion and migration during tumorigenesis. PKB activation plays a central role during epithelial-mesenchymal transition, a cellular program required for the cancer cell invasion and migration. However, the differential behavior of each PKB isoform has been shown in the regulation of epithelial-mesenchymal transition. Recent studies have suggested that PKBα (Akt1) plays a conflicting role in tumorigenesis by acting either as a pro-oncogenic factor by suppressing the apoptotic machinery or by restricting tumor invasion. PKBß (Akt2) promotes cell migration and invasion and similarly PKBγ (Akt3) has been reported to promote tumor migration. As PKB is known for its pro-oncogenic properties, it needs to be unraveled how three isoforms of PKB compensate during tumor progression. In this review, we attempted to sum up how different isoforms of PKB play a role in cancer progression, metastasis, and drug resistance.
Subject(s)
Neoplasms/enzymology , Proto-Oncogene Proteins c-akt/metabolism , Animals , Enzyme Activation , Humans , Isoenzymes , Neoplasms/pathology , Signal TransductionABSTRACT
The ethyl acetate extract from the fermentation broth of an actinomycete strain, identified as Streptomyces scabrisporus isolated from soil of Kashmir Himalayas - India, exhibited significant cytotoxic activity against a panel of human cancer cell lines. The active fraction subjected to column chromatography led to the isolation of pharmacologically potent anticancer compound whose structure was established to be alborixin on the basis of spectral data analysis. The compound exhibited antiproliferative activity against panel of cell lines N2a, MCF-7, MiaPaca-2, PC-3, HCT-116, MDA-MB-231, HL-60 and A-549 cells with IC50 of 9.7, 15.4, 7.2, 8.1, 3.2, 9.7, 7.5 and 11.5 µM respectively. Alborixin displayed the maximum cytotoxic activity against HCT-116 human colon carcinoma cells and therefore further studies were carried on this cell line. Alborixin decreased the clonogenic potential of HCT-116 cells in a dose dependent manner. It induced apoptotic cell death in HCT116 cells that were confirmed by Flow cytometric analysis of Annexin V/PI staining and microscopic examination of cellular morphology through DAPI-stained cells. Biochemical evidence of apoptosis came from elevating the intracellular ROS level that was accompanied by mitochondrial membrane potential loss, decreasing the expression profile of anti-apoptotic protein Bcl-2, whereas it augments cleavage of caspase-3 and PARP-1, activates caspase-8 and 9 with concomitant increase in expression of proapoptotic protein Bax in a dose dependent manner. These results indicate that alborixin obtained from Streptomyces scabrisporus IIIM55 induces apoptotic cell death in colon cancer cells HCT-116 and can be further evaluated for its potential as an anticancer agent.
