The present study was conducted in three parts. In the first part of this study, in vitro, antiproliferative and morphological apoptosis inducing effect of nimbolide was investigated on the selected cancer (Du-145, PC-3, EJ cell and A-549) and normal (NIH3T3 and CCD-18Co) cell lines. The cytotoxic e...
The present study was conducted in three parts. In the first part of this study, in vitro, antiproliferative and morphological apoptosis inducing effect of nimbolide was investigated on the selected cancer (Du-145, PC-3, EJ cell and A-549) and normal (NIH3T3 and CCD-18Co) cell lines. The cytotoxic effect was assayed by using MTT assay, apoptotic and necrotic cells were measured using Hoechst 33342 and propidium iodide dual staining through a fluorescent microscope and also by staining with annexin V and propidium iodide through flow cytometric analysis. The activity of caspases 3, 8 and 9 was measured by caspases colorimetric assay kits. The percentages of the viability of cells treated with different concentrations of nimbolide were significantly lower (P0.05) between treated and the non-treated cells was observed. Nimbolide exerted time and dose dependent cytotoxic effect on the cancer cell lines and mild effect on the normal cell lines. It was further confirmed through PKH 26. The number of apoptotic and necrotic cells were significantly higher in all selected cancer cell lines treated with nimbolide as compared with untreated control cells whereas in normal cell lines no significant difference was observed between nimbolide treated and untreated cells. The activity of caspases 3, 8 and 9 was also significantly higher in all cancer cell lines treated with nimbolide as compared with untreated control cells while did not change significantly in normal cell lines as compared with untreated control. It suggested that nimbolide exerted cytotoxic and apoptotic effect selectively only in cancer cells without affecting the normal cells and one of the apoptosis inducing mechanism is through the activation of caspases signaling pathways.
In the second part of this study we used methanolic extract of neem oil instead of nimbolide on the selected cancer (PC-3, DU-145 and A-549) and normal (NIH3T3 and CCD-18Co) cell lines. The cytotoxic effect induced by the neem oil extract was measured by using MTT assay and apoptotic effect of the extract was evaluated by using Hoechst 33342 and propidium iodide dual staining through a fluorescent microscope and activity of caspases 3, 8 and 9 through colorimetric assay kits. The results showed that neem oil extract significantly reduced the viability in all selected cancer cells treated with varying concentrations of extract as compared with untreated cells and less effect on normal cell lines. It also significantly increased the number of apoptotic cells, necrotic cells, and activity of caspases 3, 8 and 9 in all cancer cells treated with extract as compared with untreated cells whereas no effect on normal cell lines. It suggested that neem oil extract exerts a higher cytotoxic effect on cancer cells than normal cells and lower concentration induce apoptosis only in cancer cells and one of the apoptosis-inducing mechanism is through the activation of caspases signaling pathways. Conclusively, it implied that neem oil extract may contain one or more potential agents that can be used as a safe and effective anticancer therapy.
In the final part of this study, we investigated the antiproliferation effect and its molecular mechanism against bladder cancer EJ cells. Proliferation of EJ cells was measured by MTT assay, protein expression of cyclin dependent kinase (CDK) 2, CDK4, Cyclin D1, Cyclin E, p21, p27, p53, and phosphorylation of C-jun N terminal kinases (JNK), p38, extracellular signal regulate kinase (ERK), specific protein kinase (AKT) were analyzed by western blotting, cell migration was analyzed by scratch wound healing assay, cell invasion was assessed by Boyden chamber invasion assay, zymography assay for expression of matrix metalloproteinases (MMP)-2 and MMP-9 and electric mobility shift assay (EMSA) was performed for detection of binding activity of transcription factors, nuclear factor-kappa B (NF-kB), activator protein-1 (AP-1) and specificity protein-1 (Sp-1). The treatment of EJ cells with nimbolide inhibited cell proliferation dose dependently through down regulation of cyclin D1 and cyclin dependent kinase CDK4 without effect on cyclin E and CDK2. It also significantly upregulated the tumor suppressor protein p21 but did not effect on p27 and p53. It also induced phosphorylation of JNK and AKT as compared with control but did not effect on p38 and ERK. Furthermore, it also reduced the migration and invasion of EJ cells via inhibition of MMP-2 and MMP-9 followed by decreased binding activities of transcription factor NF-kB, AP-1 and Sp-1 motifs. Conclusively, our results showed that nimbolide had the inhibitory effect of proliferation, migration and invasion of EJ cells and may provide a new hope as a therapeutic treatment of malignancies.
The present study was conducted in three parts. In the first part of this study, in vitro, antiproliferative and morphological apoptosis inducing effect of nimbolide was investigated on the selected cancer (Du-145, PC-3, EJ cell and A-549) and normal (NIH3T3 and CCD-18Co) cell lines. The cytotoxic effect was assayed by using MTT assay, apoptotic and necrotic cells were measured using Hoechst 33342 and propidium iodide dual staining through a fluorescent microscope and also by staining with annexin V and propidium iodide through flow cytometric analysis. The activity of caspases 3, 8 and 9 was measured by caspases colorimetric assay kits. The percentages of the viability of cells treated with different concentrations of nimbolide were significantly lower (P0.05) between treated and the non-treated cells was observed. Nimbolide exerted time and dose dependent cytotoxic effect on the cancer cell lines and mild effect on the normal cell lines. It was further confirmed through PKH 26. The number of apoptotic and necrotic cells were significantly higher in all selected cancer cell lines treated with nimbolide as compared with untreated control cells whereas in normal cell lines no significant difference was observed between nimbolide treated and untreated cells. The activity of caspases 3, 8 and 9 was also significantly higher in all cancer cell lines treated with nimbolide as compared with untreated control cells while did not change significantly in normal cell lines as compared with untreated control. It suggested that nimbolide exerted cytotoxic and apoptotic effect selectively only in cancer cells without affecting the normal cells and one of the apoptosis inducing mechanism is through the activation of caspases signaling pathways.
In the second part of this study we used methanolic extract of neem oil instead of nimbolide on the selected cancer (PC-3, DU-145 and A-549) and normal (NIH3T3 and CCD-18Co) cell lines. The cytotoxic effect induced by the neem oil extract was measured by using MTT assay and apoptotic effect of the extract was evaluated by using Hoechst 33342 and propidium iodide dual staining through a fluorescent microscope and activity of caspases 3, 8 and 9 through colorimetric assay kits. The results showed that neem oil extract significantly reduced the viability in all selected cancer cells treated with varying concentrations of extract as compared with untreated cells and less effect on normal cell lines. It also significantly increased the number of apoptotic cells, necrotic cells, and activity of caspases 3, 8 and 9 in all cancer cells treated with extract as compared with untreated cells whereas no effect on normal cell lines. It suggested that neem oil extract exerts a higher cytotoxic effect on cancer cells than normal cells and lower concentration induce apoptosis only in cancer cells and one of the apoptosis-inducing mechanism is through the activation of caspases signaling pathways. Conclusively, it implied that neem oil extract may contain one or more potential agents that can be used as a safe and effective anticancer therapy.
In the final part of this study, we investigated the antiproliferation effect and its molecular mechanism against bladder cancer EJ cells. Proliferation of EJ cells was measured by MTT assay, protein expression of cyclin dependent kinase (CDK) 2, CDK4, Cyclin D1, Cyclin E, p21, p27, p53, and phosphorylation of C-jun N terminal kinases (JNK), p38, extracellular signal regulate kinase (ERK), specific protein kinase (AKT) were analyzed by western blotting, cell migration was analyzed by scratch wound healing assay, cell invasion was assessed by Boyden chamber invasion assay, zymography assay for expression of matrix metalloproteinases (MMP)-2 and MMP-9 and electric mobility shift assay (EMSA) was performed for detection of binding activity of transcription factors, nuclear factor-kappa B (NF-kB), activator protein-1 (AP-1) and specificity protein-1 (Sp-1). The treatment of EJ cells with nimbolide inhibited cell proliferation dose dependently through down regulation of cyclin D1 and cyclin dependent kinase CDK4 without effect on cyclin E and CDK2. It also significantly upregulated the tumor suppressor protein p21 but did not effect on p27 and p53. It also induced phosphorylation of JNK and AKT as compared with control but did not effect on p38 and ERK. Furthermore, it also reduced the migration and invasion of EJ cells via inhibition of MMP-2 and MMP-9 followed by decreased binding activities of transcription factor NF-kB, AP-1 and Sp-1 motifs. Conclusively, our results showed that nimbolide had the inhibitory effect of proliferation, migration and invasion of EJ cells and may provide a new hope as a therapeutic treatment of malignancies.
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