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Inhibition of phosphotidylinositol-3 kinase pathway by a novel naphthol derivative of betulinic acid induces cell cycle arrest and apoptosis in cancer cells of different origin.

Majeed R, Hamid A, Sangwan PL, Chinthakindi PK, Koul S, Rayees S, Singh G, Mondhe DM, Mintoo MJ, Singh SK, Rath SK, Saxena AK - Cell Death Dis (2014)

Bottom Line: In the present study, we have shown for the first time that HBA decreased the expression of phosphotidylinositol-3 kinase (PI3K) p110α and p85α and caused significant downregulation of pAKT and of NFκB using human leukemia and breast cancer cells as in vitro models.Also, this target-specific inhibition was associated with mitochondrial apoptosis as was reflected by the increased expression of mitochondrial bax, downregulated bcl2 and decreased mitochondrial levels of cytochrome c, together with reactive oxygen species generation and decline in mitochondrial membrane potential.Pharmacodynamic evaluation revealed that both HBA and BA were safe upto the dose of 2000 mg/kg body weight and with acceptable pharmacodynamic parameters.

View Article: PubMed Central - PubMed

Affiliation: 1] Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India [2] Bio-organic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India.

ABSTRACT
Betulinic acid (BA) is a pentacyclic triterpenoid natural product reported to inhibit cell growth in a variety of cancers. However, the further clinical development of BA got hampered because of poor solubility and pharmacological properties. Interestingly, this molecule offer several hotspots for structural modifications in order to address its associated issues. In our endeavor, we selected C-3 position for the desirable chemical modification in order to improve its cytotoxic and pharmacological potential and prepared a library of different triazoline derivatives of BA. Among them, we previously reported the identification of a potential molecule, that is, 3{1N(5-hydroxy-naphth-1yl)-1H-1,2,3-triazol-4yl}methyloxy betulinic acid (HBA) with significant inhibition of cancer cell growth and their properties. In the present study, we have shown for the first time that HBA decreased the expression of phosphotidylinositol-3 kinase (PI3K) p110α and p85α and caused significant downregulation of pAKT and of NFκB using human leukemia and breast cancer cells as in vitro models. Further it was revealed that PI3K inhibition by HBA induced cell cycle arrest via effects on different cell cycle regulatory proteins that include CDKis cyclins and pGSK3β. Also, this target-specific inhibition was associated with mitochondrial apoptosis as was reflected by the increased expression of mitochondrial bax, downregulated bcl2 and decreased mitochondrial levels of cytochrome c, together with reactive oxygen species generation and decline in mitochondrial membrane potential. The apoptotic effectors such as caspase 8, caspase 9 and caspase 3 were found to be upregulated besides DNA repair-associated enzyme, that is, PARP cleavage caused cancer cell death. Pharmacodynamic evaluation revealed that both HBA and BA were safe upto the dose of 2000 mg/kg body weight and with acceptable pharmacodynamic parameters. The in vitro data corroborated with in vivo anticancer activity wherein Ehrlich solid tumor showed that HBA as a more potent agent than BA without any body weight loss and mortality.

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Related in: MedlinePlus

(a–c) Influence of HBA on the expression of proteins involved in cell cycle regulation. HL-60, THP-1 and MCF-7 cells were treated with the indicated concentrations of HBA for 24 h. β-actin was used as an internal control to represent the same amount of proteins applied for SDS-PAGE. Specific antibodies were used for the detection of cyclins A, E and D levels. Data are representative of one of two similar experiments. ***P<0.001, **P<0.01, *P<0.05 versus control using Student's t-test. (d) Confocal immunofluorescence done on MCF-7 cells. Cells were treated with HBA and after completion of the treatment cells were incubated with primary and secondary antibodies. Nuclear staining was done with DAPI. (e–g) Effect of HBA on cell cycle phase distribution. HL-60, THP-1 and MCF-7 cells in culture were treated with the indicated concentrations of HBA for 24 h. Cells were stained with PI to determine DNA fluorescence and cell cycle phase distribution by flow cytometery as described in Materials and Methods. Fraction of cells for hypo diploid (sub-G0, ≤2n DNA) population indicative of DNA damage was analyzed from PI versus cell counts shown (%). Data are representative of one of two similar experiments. (h and i) Estimation of early and late apoptotic cell population using annexin V–FITC staining. HBA-treated HL-60, THP-1 and MCF-7 cells was analyzed using annexinV–FITC staining. Cells were incubated with the indicated concentrations of HBA for 24 h and stained with annexin V–FITC and PI to analyze apoptotic and necrotic cell populations as described in Materials and Methods. Data are representative of one of two similar experiments
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fig4: (a–c) Influence of HBA on the expression of proteins involved in cell cycle regulation. HL-60, THP-1 and MCF-7 cells were treated with the indicated concentrations of HBA for 24 h. β-actin was used as an internal control to represent the same amount of proteins applied for SDS-PAGE. Specific antibodies were used for the detection of cyclins A, E and D levels. Data are representative of one of two similar experiments. ***P<0.001, **P<0.01, *P<0.05 versus control using Student's t-test. (d) Confocal immunofluorescence done on MCF-7 cells. Cells were treated with HBA and after completion of the treatment cells were incubated with primary and secondary antibodies. Nuclear staining was done with DAPI. (e–g) Effect of HBA on cell cycle phase distribution. HL-60, THP-1 and MCF-7 cells in culture were treated with the indicated concentrations of HBA for 24 h. Cells were stained with PI to determine DNA fluorescence and cell cycle phase distribution by flow cytometery as described in Materials and Methods. Fraction of cells for hypo diploid (sub-G0, ≤2n DNA) population indicative of DNA damage was analyzed from PI versus cell counts shown (%). Data are representative of one of two similar experiments. (h and i) Estimation of early and late apoptotic cell population using annexin V–FITC staining. HBA-treated HL-60, THP-1 and MCF-7 cells was analyzed using annexinV–FITC staining. Cells were incubated with the indicated concentrations of HBA for 24 h and stained with annexin V–FITC and PI to analyze apoptotic and necrotic cell populations as described in Materials and Methods. Data are representative of one of two similar experiments

Mentions: GSK3β is a substrate of PI3K/AKT pathway that is constitutively active in unstimulated cells.13 Keeping in view the importance of GSK3β in regulation of cyclins, CDKis and thus on the cell cycle, we sought to examine the phosphorylated levels of GSK3β. We have found significant inhibition of pGSK3β in HBA-treated HL-60 cells in a dose-dependent manner (Figure 4a). In THP-1 cells, the inhibition in the pGSK3β expression was observed at slightly higher concentrations. Under similar conditions, THP-1 cells treated with BA could produce only marginal effect at higher concentration of 30 μM (Figure 4b). Looking into the expression profile of pGSK3β in MCF-7 cells, we found that HBA could inhibit this protein in a much similar manner to that observed in THP-1 cells (Figure 4c). The pGSK3β expression was also confirmed using fluorescence microscopy in MCF-7 breast cancer cells. The results clearly showed that in untreated cells pGSK3β is highly expressed in the cytoplasm after analyzing with nuclear counter stain DAPI (Figure 4d). Interestingly, HBA treatment induced a gradual decrease in the expression of pGSK3β. This inhibition of pGSK3β in response to HBA treatment coincides with its decreased phosphorylation that was observed through western blotting studies. As we have found inhibition in the expression of cyclins by treatment with HBA in all the three cancer cell lines, we wanted to study whether the treatment of HBA will affect the expression of any of the CDKis, and we observed that HBA treatment to the cancer cells of different histogenic origin caused drastic upregulation in the expression of p21 and p27 cip/kip proteins in a concentration-dependent manner (Figures 4a–c). BA the parent molecule also caused significant p21 and p27 protein upregulation at 30 μM concentration in THP-1 cells (Figure 4b). Thus inhibition in the expression of cyclins was caused due to the upregulation in the expression p21/p27 cip/kip proteins that occurred due to inhibition in the phosphorylation of pAKT and thus inhibition in the downstream effector pathways. These observations provide strong evidence that inhibition of PI3K pathway by HBA in leukemia and breast cancer cells involve regulation of cell cycle by regulating cyclins and CDKis that induce cancer cell death.


Inhibition of phosphotidylinositol-3 kinase pathway by a novel naphthol derivative of betulinic acid induces cell cycle arrest and apoptosis in cancer cells of different origin.

Majeed R, Hamid A, Sangwan PL, Chinthakindi PK, Koul S, Rayees S, Singh G, Mondhe DM, Mintoo MJ, Singh SK, Rath SK, Saxena AK - Cell Death Dis (2014)

(a–c) Influence of HBA on the expression of proteins involved in cell cycle regulation. HL-60, THP-1 and MCF-7 cells were treated with the indicated concentrations of HBA for 24 h. β-actin was used as an internal control to represent the same amount of proteins applied for SDS-PAGE. Specific antibodies were used for the detection of cyclins A, E and D levels. Data are representative of one of two similar experiments. ***P<0.001, **P<0.01, *P<0.05 versus control using Student's t-test. (d) Confocal immunofluorescence done on MCF-7 cells. Cells were treated with HBA and after completion of the treatment cells were incubated with primary and secondary antibodies. Nuclear staining was done with DAPI. (e–g) Effect of HBA on cell cycle phase distribution. HL-60, THP-1 and MCF-7 cells in culture were treated with the indicated concentrations of HBA for 24 h. Cells were stained with PI to determine DNA fluorescence and cell cycle phase distribution by flow cytometery as described in Materials and Methods. Fraction of cells for hypo diploid (sub-G0, ≤2n DNA) population indicative of DNA damage was analyzed from PI versus cell counts shown (%). Data are representative of one of two similar experiments. (h and i) Estimation of early and late apoptotic cell population using annexin V–FITC staining. HBA-treated HL-60, THP-1 and MCF-7 cells was analyzed using annexinV–FITC staining. Cells were incubated with the indicated concentrations of HBA for 24 h and stained with annexin V–FITC and PI to analyze apoptotic and necrotic cell populations as described in Materials and Methods. Data are representative of one of two similar experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4237233&req=5

fig4: (a–c) Influence of HBA on the expression of proteins involved in cell cycle regulation. HL-60, THP-1 and MCF-7 cells were treated with the indicated concentrations of HBA for 24 h. β-actin was used as an internal control to represent the same amount of proteins applied for SDS-PAGE. Specific antibodies were used for the detection of cyclins A, E and D levels. Data are representative of one of two similar experiments. ***P<0.001, **P<0.01, *P<0.05 versus control using Student's t-test. (d) Confocal immunofluorescence done on MCF-7 cells. Cells were treated with HBA and after completion of the treatment cells were incubated with primary and secondary antibodies. Nuclear staining was done with DAPI. (e–g) Effect of HBA on cell cycle phase distribution. HL-60, THP-1 and MCF-7 cells in culture were treated with the indicated concentrations of HBA for 24 h. Cells were stained with PI to determine DNA fluorescence and cell cycle phase distribution by flow cytometery as described in Materials and Methods. Fraction of cells for hypo diploid (sub-G0, ≤2n DNA) population indicative of DNA damage was analyzed from PI versus cell counts shown (%). Data are representative of one of two similar experiments. (h and i) Estimation of early and late apoptotic cell population using annexin V–FITC staining. HBA-treated HL-60, THP-1 and MCF-7 cells was analyzed using annexinV–FITC staining. Cells were incubated with the indicated concentrations of HBA for 24 h and stained with annexin V–FITC and PI to analyze apoptotic and necrotic cell populations as described in Materials and Methods. Data are representative of one of two similar experiments
Mentions: GSK3β is a substrate of PI3K/AKT pathway that is constitutively active in unstimulated cells.13 Keeping in view the importance of GSK3β in regulation of cyclins, CDKis and thus on the cell cycle, we sought to examine the phosphorylated levels of GSK3β. We have found significant inhibition of pGSK3β in HBA-treated HL-60 cells in a dose-dependent manner (Figure 4a). In THP-1 cells, the inhibition in the pGSK3β expression was observed at slightly higher concentrations. Under similar conditions, THP-1 cells treated with BA could produce only marginal effect at higher concentration of 30 μM (Figure 4b). Looking into the expression profile of pGSK3β in MCF-7 cells, we found that HBA could inhibit this protein in a much similar manner to that observed in THP-1 cells (Figure 4c). The pGSK3β expression was also confirmed using fluorescence microscopy in MCF-7 breast cancer cells. The results clearly showed that in untreated cells pGSK3β is highly expressed in the cytoplasm after analyzing with nuclear counter stain DAPI (Figure 4d). Interestingly, HBA treatment induced a gradual decrease in the expression of pGSK3β. This inhibition of pGSK3β in response to HBA treatment coincides with its decreased phosphorylation that was observed through western blotting studies. As we have found inhibition in the expression of cyclins by treatment with HBA in all the three cancer cell lines, we wanted to study whether the treatment of HBA will affect the expression of any of the CDKis, and we observed that HBA treatment to the cancer cells of different histogenic origin caused drastic upregulation in the expression of p21 and p27 cip/kip proteins in a concentration-dependent manner (Figures 4a–c). BA the parent molecule also caused significant p21 and p27 protein upregulation at 30 μM concentration in THP-1 cells (Figure 4b). Thus inhibition in the expression of cyclins was caused due to the upregulation in the expression p21/p27 cip/kip proteins that occurred due to inhibition in the phosphorylation of pAKT and thus inhibition in the downstream effector pathways. These observations provide strong evidence that inhibition of PI3K pathway by HBA in leukemia and breast cancer cells involve regulation of cell cycle by regulating cyclins and CDKis that induce cancer cell death.

Bottom Line: In the present study, we have shown for the first time that HBA decreased the expression of phosphotidylinositol-3 kinase (PI3K) p110α and p85α and caused significant downregulation of pAKT and of NFκB using human leukemia and breast cancer cells as in vitro models.Also, this target-specific inhibition was associated with mitochondrial apoptosis as was reflected by the increased expression of mitochondrial bax, downregulated bcl2 and decreased mitochondrial levels of cytochrome c, together with reactive oxygen species generation and decline in mitochondrial membrane potential.Pharmacodynamic evaluation revealed that both HBA and BA were safe upto the dose of 2000 mg/kg body weight and with acceptable pharmacodynamic parameters.

View Article: PubMed Central - PubMed

Affiliation: 1] Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India [2] Bio-organic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India.

ABSTRACT
Betulinic acid (BA) is a pentacyclic triterpenoid natural product reported to inhibit cell growth in a variety of cancers. However, the further clinical development of BA got hampered because of poor solubility and pharmacological properties. Interestingly, this molecule offer several hotspots for structural modifications in order to address its associated issues. In our endeavor, we selected C-3 position for the desirable chemical modification in order to improve its cytotoxic and pharmacological potential and prepared a library of different triazoline derivatives of BA. Among them, we previously reported the identification of a potential molecule, that is, 3{1N(5-hydroxy-naphth-1yl)-1H-1,2,3-triazol-4yl}methyloxy betulinic acid (HBA) with significant inhibition of cancer cell growth and their properties. In the present study, we have shown for the first time that HBA decreased the expression of phosphotidylinositol-3 kinase (PI3K) p110α and p85α and caused significant downregulation of pAKT and of NFκB using human leukemia and breast cancer cells as in vitro models. Further it was revealed that PI3K inhibition by HBA induced cell cycle arrest via effects on different cell cycle regulatory proteins that include CDKis cyclins and pGSK3β. Also, this target-specific inhibition was associated with mitochondrial apoptosis as was reflected by the increased expression of mitochondrial bax, downregulated bcl2 and decreased mitochondrial levels of cytochrome c, together with reactive oxygen species generation and decline in mitochondrial membrane potential. The apoptotic effectors such as caspase 8, caspase 9 and caspase 3 were found to be upregulated besides DNA repair-associated enzyme, that is, PARP cleavage caused cancer cell death. Pharmacodynamic evaluation revealed that both HBA and BA were safe upto the dose of 2000 mg/kg body weight and with acceptable pharmacodynamic parameters. The in vitro data corroborated with in vivo anticancer activity wherein Ehrlich solid tumor showed that HBA as a more potent agent than BA without any body weight loss and mortality.

Show MeSH
Related in: MedlinePlus