Effects of Payena dasyphylla (Miq.) on hyaluronidase enzyme activity and metalloproteinases protein expressions in interleukin-1beta stimulated human chondrocytes cells

Background: Hyaluronidases have been found as the target enzymes in the development of osteoarthritis (OA) disease. While there is still no curative treatment for this disease, recent studies on the treatment of OA were focused on the effectiveness of natural products which are expected to improve the symptoms with minimal side effects. The aim of this study was to screen selected Malaysian plants on their anti-hyaluronidase activity as well as to evaluate the active plant and its derived fractions on its potential anti-arthritic and antioxidant activities.Methods: A total of 20 methanolic crude extracts (bark and leaf) from ten different plants were screened using a colorimetric hyaluronidase enzymatic assay. The active plant extract (Payena dasyphylla) was then studied for its hyaluronidase inhibitory activity in the interleukin-1β (IL-1β) stimulated human chondrocytes cell line (NHAC-kn) using zymography method. The Payena dasyphylla methanolic bark extract was then fractionated into several fractions in where the ethyl acetate (EA) fraction was evaluated for its inhibitory effects on the HYAL1 and HYAL2 gene expressions using reverse transcription-polymerase chain reaction (RT-PCR) technique. While the MMP-3 and MMP-13 protein expressions were evaluated using western blot method. The phenolic and flavonoid contents of the three fractions as well as the antioxidant property of the EA fraction were also evaluated.Results: Bark extract of Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 91.63%. The plant extract also inhibited hyaluronidase expression in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml). Similarly, treatment with Payena dasyphylla ethyl acetate (EA) fraction (100 μg/ml) inhibited the HYAL1 and HYAL2 mRNA gene expressions as well as MMP-3 and MMP-13 protein expression in a dose dependent manner. Payena dasyphylla EA fraction has demonstrated the highest amount of phenolic and flavonoid content with 168.62 ± 10.93 mg GAE/g and 95.96 ± 2.96 mg RE/g respectively as compared to water and hexane fractions. In addition, the Payena dasyphylla EA fraction showed strong antioxidant activity with IC50 value of 11.64 ± 1.69 μg/mL.Conclusion: These findings have shown that Payena dasyphylla might contained potential phenolic compounds that inhibiting the key enzyme in osteoarthritis development, which is the hyaluronidase enzyme through interruption of HYAL1 and HYAL1 gene expressions. The degradation of cartilage could also be inhibited by the plant through suppression of MMP-3 and MMP-13 protein expressions. We also reported that the inhibitory effect of Payena dasyphylla on hyaluronidase activity and expression might be due to its anti-oxidant property

Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells

Hyaluronidase is one of the target enzymes in the development of osteoarthritis (OA) disease that results from the disruption of the extracellular matrix structure in the cartilage. Although various treatments for OA have been identified, they suffer from numerous drawbacks. The drawbacks are significant as OA patients require the treatment for their entire lifetime. While there is still no curative treatment for this disease, recent studies for the prevention or treatment on OA were focused on the efficacy of natural products which are capable of ameliorating the symptoms with limited side effects. Therefore, this research aimed to study the effect of Payena dasyphylla, a selected Malaysian plants on the hyaluronidases activity, and certain matrix metalloproteinases (MMPs) expression. Prior to this, a total of 20 methanolic crude extracts (bark and leaf) were screened using a colorimetric anti-hyaluronidase enzymatic assay. Out of the plants tested, bark extracts of Palaquium gutta, Pauteria obovatta and Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 88.82 ± 0.15, 90.47 ± 0.09 and 91.63 ± 0.21 percent respectively. Due to insufficient amount of samples, only Payena dasyphylla with an IC50 value of 48.75 ± 8.97 μg/ml was further studied for its effect on hyaluronidase activity in interleukin-1β (100 ng/ml) stimulated human chondrocytes cells (NHAC-kn) using the zymography method. Payena dasyphylla was then fractionated into aqueous (Aq), ethyl acetate (EA) and hexane fractions where the EA fraction which showed the highest inhibitory activity against hyaluronidase was selected for further evaluation on its capability inhibiting the gene expressions of HYAL1 and HYAL2 using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technique. Besides evaluating the effect of Payena dasyphylla on the hyaluronidases, this plant extract was also tested on the expression of other mediators mainly on MMP-3 and MMP-13 using Western blotting method. Payena dasyphylla was then further investigated for its total phenolic and flavonoid content as well as antioxidant activity. The antioxidant activity was measured using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay whereas total phenolic content was determined using the Folin-ciocalteu’s method. Results showed that Payena dasyphylla methanolic extract (100 μg/ml) inhibited hyaluronidase activity in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml) stimulation. Similarly, treatment with Payena dasyphylla EA fraction (100 μg/ml) also inhibited the HYAL1 and HYAL2 gene expressions. Cytotoxicity results revealed that both Payena dasyphylla methanolic and EA fraction did not show any concomitant effect on the NHAC-kn cell line at all concentrations tested. On the other hand, the Payena dasyphylla EA fraction also inhibited MMP-3 and MMP-13 protein expression. Interestingly, treatment without IL-1β (10 ng/ml) stimulation revealed a significant difference as MMP-3 and MMP-13 protein expressions were seen only with the presence of IL-1β inducer. In addition, Payena dasyphylla EA fraction showed the highest amount of phenolic and flavonoid content with 168.62 ± 10.93 mg GAE/g and 95.96 ± 2.96 mg RE/g respectively compared to aqueous and hexane fractions. Besides, Payena dasyphylla EA extract exhibited antioxidant activity of 82.19% at the concentration of 100 μg/ml with IC50 value of 14.10 ± 1.11 μg/ml. As a conclusion, these findings showed that Payena dasyphylla contained potential classes of compound inhibiting hyaluronidases, HYAL1 and HYAL2 gene expressions as well as MMP-3 and MMP-13 protein expressions that are responsible for the degeneration of the cartilage tissue. Therefore, this plant might serve as an alternative in formulating new generation of drugs in the management of cartilage damage

Anticancer activity and Molecular mechanisms of a selected diarylpentanoid treated on androgen-independent metastatic human prostate cancer cells

Prostate cancer remains incurable, highlighting a need for alternative treatment methods using natural sources. Specific structural modification on curcumin, an active ingredient of the spice turmeric to produce curcumin diarylpentanoid demonstrated improved anticancer activity on prostate cancer cells; evident by its significant growth suppressive effect and cancer killing potential. Investigation on gene and protein expression study provided an understanding into the potential anticancer activity of the compound. Overall, curcumin diarylpentanoid demonstrated potential anticancer activity and warrants further research for its use in the treatment of prostate cancer

Identification of commonly regulated protein targets and molecular pathways in PC-3 and DU145 androgen-independent human prostate cancer cells treated with the curcumin analogue 1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one

Objective: To identify mutually regulated proteins in PC-3 and DU145 androgen-independent prostate cancer cell lines treated with 1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one (MS17), and to study the molecular pathways that contributed to the anticancer activity of MS17. Methods: PC-3 and DU145 cells were treated with 3 × EC50 (15 μM) concentration of MS17 for 24 h and were subjected to protein expression profiling using two-dimensional gel electrophoresis and protein identification by mass spectrometry. Selected differentially expressed proteins with significant P-value of P<0.05 and fold change over 1.5-folds were filtered through and ontologically classified. Mutually regulated proteins were ranked by fold change and identified as common protein targets of MS17. Results: Profiling data revealed that, the mutually down-regulated proteins included ACTB and ACTG associated with structural molecule activity, ACTN1 with cell cycle, ACTN4 with cell migration, HNRPK with apoptosis, PLST with morphogenesis and TERA with proteolysis. However, the expressions of CH60 and HS71A respectively associated with response to unfolded protein demonstrated opposing regulation in PC-3 and DU145 cells. Pathway analysis of the differentially expressed proteins in PC-3 cells demonstrated the modulation of top pathways associated with cell-cell adhesion and cytoskeletal organization while in DU145 cells the pathways were associated with proteosomal degradation, regulation of electrolytes and water, regulation control of germ cells and organization of filament assembly/disassembly. Conclusions: The findings of the present study provide an understanding on the anti-tumorigenic activity of MS17 at the proteome level and warrant further research for its potential application for the management and treatment of androgen-independent prostate cancer

Anti-proliferative effect and induction of apoptosis in androgen-independent human prostate cancer cells by 1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one

Curcumin has poor in vivo absorption and bioavailability, highlighting a need for new curcumin analogues with better characteristics in these aspects. The aim of this study is to determine the anti-cancer properties of four selected curcumin analogues, on the cytotoxicity, proliferative and apoptotic effects on androgen-independent human prostate cancer cells (PC-3 and DU 145). Initial cytotoxicity screening showed MS17 has the highest cell inhibitory effect, with EC50 values of 4.4 ± 0.3 and 4.1 ± 0.8 µM, followed by MS13 (7.5 ± 0.1 and 7.4 ± 2.6 µM), MS49 (14.5 ± 1.2 and 12.3 ± 2.3 µM) and MS40E (28.0 ± 7.8 and 30.3 ± 1.9 µM) for PC-3 and DU 145 cells, respectively. Time-dependent analysis also revealed that MS13 and MS17 displayed a greater anti-proliferative effect than the other compounds. MS17 was chosen based on the high selectivity index value for further analysis on the morphological and biochemical hallmarks of apoptosis. Fluorescence microscopy analysis revealed apoptotic changes in both treated prostate cancer cells. Relative caspase-3 activity increased significantly at 48 h in PC-3 and 12 h in DU 145 cells. Highest enrichment of free nucleosomes was noted at 48 h after treatment with MS17. In conclusion, MS17 demonstrated anti-proliferative effect and induces apoptosis in a time and dose-dependent manner suggesting its potential for development as an anti-cancer agent for androgen-independent prostate cancer