Proteomic Analysis of HepG2 Cells Reveals FAT10 and BAG2 Signaling Pathways Affected by a Protease Inhibitor from Tinospora cordifolia (Willd.) Hook. f. and Thoms Stem. Extract Among the Different Plant and Microbial Samples Analyzed.

Objectives: Dysregulation of proteolysis underlies diseases like cancer. Protease inhibitors (PIs) regulate many biological functions and hence have potential anticancer properties. With this background, the current study aimed to identify the PI from natural sources such as plants and microbes against trypsin (a protease), which was assayed against casein, using an ultraviolet spectrophotometer-based methodology. Materials and Methods: PI extracted from a few plants and microbial samples were screened for their PI activity against trypsin. The PI from the most promising source in our study, Tinospora cordifolia (Willd.) Hook. f. and Thoms. stem, was partially purified using ammonium sulfate precipitation followed by dialysis. The PI activity of the partially purified inhibitor was analyzed against chymotrypsin and collagenase enzymes, and the cytotoxic effect of the PI was checked on HepG2 (liver carcinoma) cells by MTT- [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]- assay. Liquid Chromatograography Mass Spectrometry -based proteomic studies were performed on HepG2 cells to understand the signaling pathways affected by the PIs in the liver cancer cell line. Results: Among the samples tested the PIs from T. cordifolia stem extract had the highest inhibitory activity (72.0%) against trypsin along with cytotoxicity to HepG2 cells. After partial purification by 80.0% ammonium sulfate precipitation, PI had increased inhibitory activity (83.0%) against trypsin and enhanced cytotoxicity (47.0%) to HepG2 cells. Proteomic analysis of the PI-treated HepG2 cells revealed that BAG2 and FAT10 signaling pathways were affected, which may have caused the inhibition of cancer cell proliferation. Conclusion: PI from T. cordifolia stem has promising anticancer potential and hence can be used for further purification and characterization studies toward cancer drug development.

Analysis of the Anticancer Mechanism of OR3 Pigment from Streptomyces coelicolor JUACT03 Against the Human Hepatoma Cell Line Using a Proteomic Approach.

This study assessed OR3 pigment, derived from Streptomyces coelicolor JUACT03, for its anticancer potential on HepG2 liver cancer cells and its safety on HEK293 normal cells. OR3 induced apoptosis and inhibited HepG2 cell proliferation, confirmed by caspase activation, Sub-G1 phase cell cycle arrest, and reduced colony formation. Proteomic analysis revealed altered expression of proteins associated with ribosomal function, mRNA processing, nuclear transport, proteasome activity, carbohydrate metabolism, chaperone function, histone regulation, and vesicle-mediated transport. Downregulation of proteins in MAPKAP kinase1, EIF2, mTOR, and EIF4 pathways contributed to apoptosis and cell cycle arrest. Changes in c-MYC, FUBP1 target proteins and upregulation of Prohibitin-1 (PHB1) were also noted. Western blot analysis supported alterations in eIF2, mTOR, and RAN pathways, including downregulation of RAB 5, c-MYC, p38, MAPK1, and MAPK3. OR3 exhibited significant anti-angiogenic activity in the in ovo CAM assay. In summary, OR3 demonstrated strong anticancer effects, inducing apoptosis, hindering proliferation, and displaying antiangiogenic properties. These findings highlight OR3's potential as an anticancer drug candidate, warranting further in vivo exploration.

Proteomic Analysis of Human Breast Cancer MCF-7 Cells to Identify Cellular Targets of the Anticancer Pigment OR3 from Streptomyces coelicolor JUACT03.

Search for ideal compounds with known pathways of anticancer mechanism is still a priority research focus for cancer, as it continues to be a major health challenge across the globe. Hence, in the present study, anticancer potential of a yellow pigment fraction, OR3, isolated from Streptomyces coelicolor JUACT03 was assessed on the breast cancer cell line MCF-7. TLC-fractionated OR3 pigment was subjected to HPLC and GC-MS analysis for characterization and identification of the bioactive component. MCF-7 cells were treated with IC50 concentration of OR3 and the molecular alterations were analyzed using mass spectrometry-based quantitative proteomic analysis. Bioinformatics tools such as STRING analysis and Ingenuity Pathway Analysis were performed to analyze proteomics data and to identify dysregulated signaling pathways. As per our obtained data, OR3 treatment decreased cell proliferation and induced apoptotic cell death due to significant dysregulation of protein expressions in MCF-7 cells. Altered expression included the ribosomal, mRNA processing and vesicle-mediated transport proteins as a result of OR3 treatment. Downregulation of MAPK proteins, NFkB, and estradiol signaling was identified in OR3-treated MCF-7 cells. Mainly eIF2, mTOR, and eIF4 signaling pathways were altered in OR3-treated cells. GC-MS data indicated the presence of novel compounds in OR3 fraction. It can be concluded that OR3 exhibits potent anticancer activity on the breast cancer cells mainly through altering the expression and affecting the signaling proteins which are involved in different cell proliferation/apoptotic pathways thereby causing inhibition of cancer cell proliferation, survival and metastasis.

A combination of semi-purified L-methioninase with tamoxifen citrate to ameliorate breast cancer in athymic nude mice.

BACKGROUND: Chemotherapy nonspecifically targets both tumor and healthy proliferating cells. Methionine deprivation using L-methioninase along with chemotherapy appears promising towards cancer management. The present study is an attempt to use a new combination of L-methioninase with Tamoxifen (TAM) to treat breast cancer in mice. METHODS AND RESULTS: L-Methioninase from Methylobacterium sp. was partially purified (SPMet's) by cold acetone precipitation and lyophilized. Its cytotoxicity effect, alone and in combination with Tamoxifen, was evaluated in vitro (MCF-7) cells and in vivo (athymic nude mice) conditions. SPMet's was found to inhibit the growth of MCF-7 cells with an IC50 value of 47.05 µg/ml, while the combination of SPMet's and TAM had an IC50 of 6.4 µg/ml. Athymic nude mice were grouped into: Group-I - Tumor control; Group-II - TAM; Group-III - SPMet's; Group-IV - SPMet's + TAM. Tumor growth inhibition (TGI) was maximum in Group-IV with 84.65% followed by Group-II with 65.12%. Hematological and Biochemical parameters in Group-II, III, and IV were restored to normal levels. Tumor histopathology showed increased apoptosis and necrosis in Group-IV. Caspases 3 & 8 gene upregulation was significantly higher in Group-IV than other treated groups, indicating higher efficacy of the combination approach. CONCLUSION: This is the first study report about a combination of SPMet's and TAM on in vivo breast cancer model, with significantly higher anticancer activity and without noticeable side effects. The findings of this study have several important implications for future clinical studies.

Modulation of Bax and Bcl-2 genes by secondary metabolites produced by Penicillium rubens JGIPR9 causes the apoptosis of cancer cell lines.

Search for an efficient anti-cancer compound of natural origin with well-defined mechanisms of action is an important scientific pursuit today, due to cancer being the second leading cause for the death of affected people. The members of the genus Penicillium are one of the important sources of bioactive compounds. In the present study, Penicillium rubens, isolated from a garden soil in Madurai district of Tamil Nadu, was found to produce a highly promising anti-cancer metabolite. The percentage viabilities of HepG2, HeLa and MCF-7 cancer cells treated with the bioactive fraction (P5) isolated from P. rubens, ranged between 40-50% after 96 h. Apoptosis induction was found to be the major reason for the observed reduction in cancer cell proliferation and cell count which was confirmed by caspase activity, DNA fragmentation, clonogenic assay, cell cycle analysis and LDH assays. The upregulation of proapoptotic Bax, coupled with the downregulation of anti-apoptotic Bcl-2 expressions were confirmed by RT-qPCR and flow cytometry methods. The current study also indicated an upregulation of p53 which further strengthened the apoptogenic property of P5 fraction. Non-toxicity of P5 was demonstrated on normal peripheral lymphocytes. The analysis of P5 fraction through GC-MS indicated the presence of indole-2, 3-(4,4-dimethyl-3-thiosemicarbazone) as one of the major compounds.