AIM OF THE STUDY: This study was aimed to reveal three different PBs' aqueous extracts(viz. PB-A, PB-B, PB-C) chemical constituent's profile using GC-MS analysis, anticancer property on A375, HeLa and MCF7 cancer cells, toxicity profile on zebrafish embryo morphology, EC50, LC50 and teratogenicity index.
MATERIALS AND METHODS: PBs' extracts characterization was performed through GC-MS analysis, in vitro anticancer effect was carried out on A375, HeLa and MCF7 cancer cell lines and finally and toxicity properties on three different PBs aqueous extracts (viz. PB-A, PB-B, PB-C) were determined using zebrafish embryo model.
RESULTS: The GC-MS analysis revealed 10 similar compounds in all PBs' extracts. Dilauryl thiodipropionate was found to be a major compound in all PBs' extracts followed by tetradecanoic acid. An in vitro anticancer study revealed PB extracts exerted median inhibition concentration (IC50) <50 μg/mL, on cancer cells viz. A375, HeLa and MCF7 with no significant toxicity on normal cells viz. NHDF cells. In vivo toxicity of PBs extracts found affecting tail detachment, hatching, craniofacial, brain morphology, soft tissues, edema, spinal, somites, notochord and cardiovascular system (brachycardia, disruption of blood circulation) deformities. The LC50 and EC50 demonstrated PB extracts effect as dose and time dependent with median concentration <150.0 μg/mL. Additionally, teratogenicity index (TI) viz. >1.0 revealed teratogenic property for PB extracts.
CONCLUSIONS: The findings revealed that all three PBs aqueous extracts possessed anticancer activity and exhibited significant toxicological effects on zebrafish embryos with high teratogenicity index. Hence, its use as an anticancer agent requires further investigation and medical attentions to determine its safe dose.
METHODS: PNMA5 mutants were generated through deletion or site-directed mutagenesis and transiently expressed in human cancer cell lines to investigate their role in apoptosis, subcellular localization, and potential interaction with MOAP-1 through apoptosis assays, fluorescence microscopy, and co-immunoprecipitation studies, respectively.
RESULTS: Over-expressed human PNMA5 exhibited nuclear localization pattern in both MCF-7 and HeLa cells. Deletion mapping and mutagenesis studies showed that C-terminus of PNMA5 is responsible for nuclear localization, while the amino acid residues (391KRRR) within the C-terminus of PNMA5 are required for nuclear targeting. Deletion mapping and co-immunoprecipitation studies showed that PNMA5 interacts with MOAP-1 and N-terminal domain of PNMA5 is required for interaction with MOAP-1. Furthermore, co-expression of PNMA5 and MOAP-1 in MCF-7 cells significantly enhanced chemo-sensitivity of MCF-7 to Etoposide treatment, indicating that PNMA5 and MOAP-1 interact synergistically to promote apoptotic signaling in MCF-7 cells.
CONCLUSIONS: Our results show that PNMA5 promotes apoptosis signaling in HeLa and MCF-7 cells and interacts synergistically with MOAP-1 through its N-terminal domain to promote apoptosis and chemo-sensitivity in human cancer cells. The C-terminal domain of PNMA5 is required for nuclear localization; however, both N-and C-terminal domains of PNMA5 appear to be required for pro-apoptotic function.