Effect of CB2 Stimulation on Gene Expression in Pediatric B-Acute Lymphoblastic Leukemia: New Possible Targets

Abstract

Acute lymphoblastic leukemia type B (B-ALL) is the most common kind of pediatric leukemia, characterized by the clonal proliferation of type B lymphoid stem cells. Important progress in ALL treatments led to improvements in long-term survival; nevertheless, many adverse long-term consequences still concern the medical community. Molecular and cellular target therapies, together with immunotherapy, are promising strategies to overcome these concerns. Cannabinoids, enzymes involved in their metabolism, and cannabinoid receptors type 1 (CB1) and type 2 (CB2) constitute the endocannabinoid system, involved in inflammation, immune response, and cancer. CB2 receptor stimulation exerts anti-proliferative and anti-invasive effects in many tumors. In this study, we evaluated the effects of CB2 stimulation on B-ALL cell lines, SUP-B15, by RNA sequencing, Western blotting, and ELISA. We observe a lower expression of CB2 in SUP-B15 cells compared to lymphocytes from healthy subjects, hypothesizing its involvement in B-ALL pathogenesis. CB2 stimulation reduces the expression of CD9, SEC61G, TBX21, and TMSB4X genes involved in tumor growth and progression, and also negatively affects downstream intracellular pathways. Our findings suggest an antitumor role of CB2 stimulation in B-ALL, and highlight a functional correlation between CB2 receptors and specific anti-tumoral pathways, even though further investigations are needed.

Keywords: CB2 receptors; RNA sequencing; SUP-B15 cell line; acute lymphoblastic leukemia; endocannabinoid system.

Figure 1

CB2 gene (A) and protein (B) expression levels in SUP-B15 cell line compared with CTR lymphocytes. For real-time PCR (A), CB2 mRNA expression levels of SUP-B15 cell line are compared with healthy controls-derived lymphocytes (CTR). Results are normalized for the housekeeping gene β-actin, and shown as mean ± S.D. of three independent experiments * indicates p ≤ 0.05 compared to CTR. Western blot (B) was performed starting from 20 μg of total lysates. The most representative images are displayed. The proteins were detected using Image Studio Digits software, and the intensity of immunoblots compared to the control, taken as 1 (arbitrary unit), were quantified after normalizing with respective loading controls for the housekeeping protein β-actin. Histogram shows CB2 receptor expression levels as the mean ± S.D. of three independent experiments. A t-test was used for statistical analysis. * indicates p ≤ 0.05 compared to CTR.

Figure 2

RAC-1 (A), pAKT (B), and MMP-2 (D) protein expression levels in SUP-B15 cell line after treatment with JWH-133 and AM630 compared with non-treated control (NT), determined by Western blot, starting from 20 μg of total lysates. The most representative images are displayed. The proteins were detected using Image Studio Digits software, and the intensity of immunoblots compared to the control, taken as 1 (arbitrary unit), were quantified after normalizing with respective loading controls for the housekeeping protein β-actin. Histogram shows RAC-1, pAKT, and MMP-2 protein expression levels as the mean ± S.D. of three independent experiments. A t-test was used for statistical analysis. * indicates p ≤ 0.05 compared to NT. (C) IFN-γ concentrations (pg/mL) in SUP-B15 cells after treatment with JWH-133 and AM630 compared with non-treated control (NT), determined by enzyme-linked immunosorbent assay (ELISA Assay). Histogram shows IFN-γ concentrations as the mean ± S.D. of three independent experiments. The cytokine concentration was determined on a standard concentration curve, according to the manufacturer’s instructions. A t-test was used for statistical analysis. * indicates p ≤ 0.05 compared to NT.