BH3-only protein Bik is involved in both apoptosis induction and sensitivity to oxidative stress in multiple myeloma

Abstract

Background: although gene expression profile of multiple myeloma (MM) patients shows a wide range of Bik/Nbk expression, varying from absent to high, its regulation and function in myeloma cells is poorly understood. Thus, we addressed these questions in MM.

Methods: human myeloma cell lines (HMCLs) and primary purified myeloma cells were studied for Bcl-2 family protein expression by western blot and further correlation analysis was performed. Correlative study between Bik and thyrotroph embryonic factor (TEF) transcription factor expression was analysed by PCR. Stress oxidative response was analysed by flow cytometry.

Results: a strong expression of Bik protein was found only in one out of three of HMCL and correlated to Bcl-2 expression (P=0.0006). We demonstrated that Bik could be regulated at the protein level by Bcl-2 and at the transcriptional level by TEF. Bik overexpression sensitises myeloma cells to oxidative stress whereas Bik silencing increases resistance to H(2)O(2) oxidative stress. Furthermore, Bik ectopic expression disrupts Bim/Bcl-2 and Bim/Bcl-xL endogenous complexes triggering Bim release that could induce Bax and Bak activation.

Conclusions: ours results suggest that Bik has a role in both, apoptosis induction and sensitivity to oxidative stress in myeloma cells. Small BH3 mimetic molecules should be considered for further apoptosis-based therapy in myeloma cells expressing endogenous Bik/Bcl-2 complexes.

Figure 1

Bik expression is correlated with Bcl-2 expression. (A) Expression of BH3-only pro-apoptotic (Bik, Bim, Puma and Noxa) and antiapoptotic (Bcl-2, Mcl-1, Bcl-x_L) proteins on HMCL. Equivalent amounts of cell lysates (70 μg of protein) were subjected to immunodetection with the indicated antibodies. Protein loading was controlled by probing with an anti-actin antibody. (B) Expression of Bik and Bcl-2 proteins on purified primary myeloma cells. (C) Bik and Bcl-2 from HMCL were quantified using Image-J software, both proteins were normalised by actin.

Figure 2

Bcl-2 expression stabilises Bik protein. (A) Bik and Bcl-2 form endogenous complexes in XG-2 and KMS-12PE HMCL. Lysates were subjected to immunoprecipitations with anti-Bcl-2 or anti-Mcl-1 antibodies and immunoblotting for the indicated proteins. (B) Ectopic expression of Bcl-2 stabilises Bik protein. Cells were transfected with pRcCMV-Bcl-2 or an empty vector. At 48 h after transfection, cells were harversted. Extracts were then immunoblotted for the indicated proteins. (C) Bcl-2 silencing downregulates Bik expression. U266 cells were transfected with control or Bcl-2 siRNA, protein expression was determined 24 h after transfection.

Figure 3

TEF transcription factor expression is correlated to Bik levels. (A)The mRNA levels of TEF and Bik of 24 HMCL were analysed by RT–PCR. Actin mRNA was used as an amplification control. Wild-type p53 HMCL are marked by ^*. (B) U266 and NCI-H929 cell lines were transfected with non-target control or TEF-specific siRNA. The mRNA levels were analysed by RT–PCR 72 h after transfection.

Figure 4

Bik contributes to the oxidative stress response. (A) Transient expression of Bik sensitises KMM-1 to oxidative stress. Cell line KMM-1 was transiently transfected with Bik complementary DNA or an empty vector (control), after 48 h, expression levels of Bik protein were determined by western blot. Transfected cells were treated with 200 μM H₂O₂ and then ROS generation was measured by dihydroethidine (DHE) staining followed by fluorescence-activated cell sorting analysis. (B) Bik silencing promotes protection to oxidative stress. U266 were transfected with non-target control or Bik-specific siRNA, 24 h after transfection, lysates were obtained and analysed for Bik expression levels. Transfected cells were treated with 200 μM H₂O₂ and stained as above. Results are representative of three independent experiments.

Figure 5

Bik overexpression induces cell death in myeloma cells. (A) KMM-1 cells were transfected either with empty vector (thin line) or Bik (thick line) complementary DNA, after 48 h cell death was measured by Apo2.7 staining. Results are representative of three independent experiments. (B) Cell lysates were analysed by western blotting analysis to assess caspase-9 and caspase-3 activation. (C) Cells were co-transfected with Bik cDNA in the presence of control, Bax, Bak or Bax and Bak siRNA. At 48 h after transfection, cells were analysed for Apo2.7 staining. Expression levels of Bax and Bak were determined by western blot. (D) KMM-1 cells were transfected either with empty vector or Bik cDNA, 24 h after transfection cells were harvested and cell lysates were obtained. Bik, Bcl-2 and Bcl-xL immunoprecipitates were analysed by western blot.