Changes in expression of these components were quantified, and the findings are summarized in Figure 4C. Figure 4 Expression of proteins associated with the PI3K/Akt signaling and the intrinsic (mitochondrial) apoptotic pathways after varying times of treatment of CA46 cells with baicalin. (A) Expression of p-Akt in various untreated cell types as detected by phospho-Akt specific antibody. Lane 1, CA46 cells;
lane 2, Jurkat cells; lane 3, K562 cells; lane 4, HL-60 cells; lane 5, normal peripheral blood mononuclear cells-1; lane 6, normal peripheral blood Acalabrutinib nmr mononuclear cells-2. (B-E) CA46 cells were treated with 40 μM baicalin for the times indicated. Protein expression was analyzed by Western blotting. (B) Western
blot showing expression of β-actin, Akt, p-Akt, NF-κB, IκB, p-IκB, mTOR and p-mTOR. (C) Expression of p-Akt/Akt, NF-κB, IκB, p-IκB, and p-mTOR/mTOR relative to that of β-actin. (D) Western blot showing expression of β-actin, Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3, and uncleaved (116 kD) and cleaved (85 kD) PARP. (E) Expression of cleaved caspase-9, cleaved caspase-3, uncleaved and cleaved PARP, Bax, and Bcl-2 relative to that of β-actin. Findings are representative of those obtained on three separate occasions. *P <0.05 compared to the 0 h control; † P <0.05 compared to 24 h treatment; ‡ P <0.05 compared to 48 h treatment. 5-Fluoracil clinical trial The profound decreases in expression of total cellular NF-kB and p-IkB, accompanied by significant increases in IkB expression, in response to baicalin treatment were interpreted to indicate a condition wherein nuclear NF-kB signaling should be dramatically impaired. Accordingly, expression of nuclear NF-kB was reduced by 25.8%, 50.4% and 65.4% at 24, 48 and 72 h of treatment with 40 μM baicalin, respectively Phenylethanolamine N-methyltransferase (not shown). Activation of
the intrinsic mitochondrial apoptotic pathway It was considered essential to ascertain whether baicalin suppresses proliferation of CA46 cells and promotes DNA fragmentation in these cells through activation of the intrinsic (mitochondrial) apoptotic pathway. To this end, expression of relevant apoptosis-related proteins was examined by Western blotting. Treatment with baicalin increased expression of the pro-apoptotic proteins Bax, activated (cleaved) caspase 3, activated (cleaved) caspase 9, and activated (cleaved) PARP. By contrast, expression of the anti-apoptotic protein Bcl-2 and of the inactive form of PARP was decreased following treatment with the drug (Figure 4D). Relative expression of these proteins after baicalin treatment was quantified, and findings are presented in Figure 4E.