![Figure 5. SMO inhibitor enhanced the cytotoxic effect of NF-κB inhibitor on DLBCL cells. (A) BJAB and DOHH2 were treated with cyclopamine-KAAD and/or BAY11-7082. Combined treatments resulted in enhanced decrease of cell viability, as determined by MTS assays (at 48 hours), compared with treatment with the BAY11-7082 alone. (B) Annexin V and PI plots showed the percentage of apoptosis at 48 hours baseline (dimethylsulfoxide [DMSO], a), after treating with cyclopamine-KAAD or BAY11-7082 alone (b-c), and after combination of both inhibitors (d). The percentage of apoptosis was measured 48 hours after treatments and determined by Annexin V and PI assays. Data are presented as mean ± standard deviation (SD) of 3 experiments. *P < .05. (C) Western blot analysis showed that combining cyclopamine-KAAD with BAY11-7082 resulted in an increase of cleaved poly (adenosine 5′-diphosphate-ribose) polymerase 1 and a decrease of p-p65 compared with that treated with BAY11-7082 alone. (D) Quantitative real-time PCR showed that combined treatments resulted in marked decrease in the mRNA levels of GLI1, BCL2, and BCL-xL compared with BAY11-7082 treatment alone. These results were normalized to 18S mRNA level and expressed as fold changes in mRNA expression compared with control. Data are presented as mean ± SD of 3 experiments. *P < .05. (E) Proposed working model for SMO-mediated NF-κB activation using a GPCR signal transduction pathway. In the presence of Hh ligands, SMO is released from PTCH1 inhibition and gets activated. Activated SMO transduces signals through a canonical Hh pathway (GLI transcription factors) as well as through noncanonical a Hh pathway by recruiting and activating Gαi and Gα12. As a consequence, the Gβγ subunit is released and may contribute to phosphorylate and activate PKC-β. Gα12 may also contribute to the activation of PKC-β through the ρ family of small GTPases. Activated PKC-β phosphorylates and activates CARMA1 leading to the assembly of the CBM signalosome, polyubiquitination of TRAF6 and NEMO at K63, and followed by degradation of IκBα and activation of NF-κB pathway.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/23/10.1182_blood-2012-12-470153/4/4718f5.jpeg?Expires=1735565378&Signature=NYzLxUC1lLSLitgeHi5GkyzFNZ-5-Idr21O3X8qT0Sts0hsxbZHDYofvBlzPYj9fVKldBZsgY9d-N7WTbows41ned4LZiHUUuqJmb-dGjwN2Q6FexzfbyAdDob2HTuPpiT3wQy~R1Qw8WX5~NcPQXW5urYBqFzRZNvKTBdcBuYPoJwSlYn0KnojaQxFRxlcvAStQhfXt7MSHDcWkMx8Xj5RfwAJ-ibLOB5-AMaz0Zdis8yMgZ2YpHWuZDdECliSPgu-vO-uatNfnF7R9lIqfSvcQ~O~iLxqeKt~9K3~peEyQZxcyWgFo0Xc-hSFa7e09XGTTkO4yFSRQpy0HvI8~zQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
SMO inhibitor enhanced the cytotoxic effect of NF-κB inhibitor on DLBCL cells. (A) BJAB and DOHH2 were treated with cyclopamine-KAAD and/or BAY11-7082. Combined treatments resulted in enhanced decrease of cell viability, as determined by MTS assays (at 48 hours), compared with treatment with the BAY11-7082 alone. (B) Annexin V and PI plots showed the percentage of apoptosis at 48 hours baseline (dimethylsulfoxide [DMSO], a), after treating with cyclopamine-KAAD or BAY11-7082 alone (b-c), and after combination of both inhibitors (d). The percentage of apoptosis was measured 48 hours after treatments and determined by Annexin V and PI assays. Data are presented as mean ± standard deviation (SD) of 3 experiments. *P < .05. (C) Western blot analysis showed that combining cyclopamine-KAAD with BAY11-7082 resulted in an increase of cleaved poly (adenosine 5′-diphosphate-ribose) polymerase 1 and a decrease of p-p65 compared with that treated with BAY11-7082 alone. (D) Quantitative real-time PCR showed that combined treatments resulted in marked decrease in the mRNA levels of GLI1, BCL2, and BCL-xL compared with BAY11-7082 treatment alone. These results were normalized to 18S mRNA level and expressed as fold changes in mRNA expression compared with control. Data are presented as mean ± SD of 3 experiments. *P < .05. (E) Proposed working model for SMO-mediated NF-κB activation using a GPCR signal transduction pathway. In the presence of Hh ligands, SMO is released from PTCH1 inhibition and gets activated. Activated SMO transduces signals through a canonical Hh pathway (GLI transcription factors) as well as through noncanonical a Hh pathway by recruiting and activating Gαi and Gα12. As a consequence, the Gβγ subunit is released and may contribute to phosphorylate and activate PKC-β. Gα12 may also contribute to the activation of PKC-β through the ρ family of small GTPases. Activated PKC-β phosphorylates and activates CARMA1 leading to the assembly of the CBM signalosome, polyubiquitination of TRAF6 and NEMO at K63, and followed by degradation of IκBα and activation of NF-κB pathway.
