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. 2022 Nov;12(11):2762-2777.
doi: 10.1007/s13346-022-01137-2. Epub 2022 Feb 25.

Anticancer and chemosensitization effects of cannabidiol in 2D and 3D cultures of TNBC: involvement of GADD45α, integrin-α5, -β5, -β1, and autophagy

Sunil Kumar Surapaneni #  1 , Nilkumar Patel #  1 , Li Sun  2 , Nagavendra Kommineni  1 , Anil Kumar Kalvala  1 , Aragaw Gebeyehu  1 , Peggy Arthur  1 , Leanne C Duke  2 , Ramesh Nimma  1 , David G Meckes Jr  2 , Mandip Singh  3
Affiliations

Anticancer and chemosensitization effects of cannabidiol in 2D and 3D cultures of TNBC: involvement of GADD45α, integrin-α5, -β5, -β1, and autophagy

Sunil Kumar Surapaneni et al. Drug Deliv Transl Res. 2022 Nov.

Abstract

To date, promising therapy for triple negative breast cancer (TNBC) remains a serious concern clinically because of poor prognosis, resistance, and recurrence. Herein, anti-cancer potential of synthetic cannabidiol (CBD; Purisys, GA; GMP grade) was explored either alone or as a chemosensitizer followed by post-treatment with doxorubicin (DOX) in TNBC (i.e., MDA-MB-231 and MDA-MB-468) cells. In comparison to 2D cultures, CBD showed greater IC50 values in 3D (LDP2 hydrogel based) cultures of MDA-MB-231 (6.26-fold higher) and MDA-MB-468 (10.22-fold higher) cells. Next-generation RNA sequencing revealed GADD45A, GADD45G, FASN, LOX, and integrin (i.e., -α5, -β5) genes to be novelly altered by CBD in MDA-MB-231 cells. CIM-16 plate-based migration assay and western blotting disclosed that CBD induces anti-migratory effects in TNBC cells by decreasing fibronectin, vimentin, and integrins-α5, -β5, and -β1. Western blotting, RT-qPCR, and immunocytochemistry revealed that CBD inhibited autophagy (decreased Beclin1, and ATG-5, -7, and -16) of TNBC cells. CBD pre-treatment increased DOX sensitivity in TNBC cells. CBD pre-treatment accompanied by DOX treatment decreased LOX and integrin-α5, and increased caspase 9 protein respectively in MDA-MB-468 cells.

Keywords: Autophagy; Cannabidiol; Doxorubicin; GADD45α; Integrins; TNBC.

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Conflict of interest statement

Competing interests The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Anti-cancer efficacy of cannabidiol and doxorubicin. A, B, C 2D cultures of TNBC and MCF-10A cells; D 3D cultures of TNBC cells and 3D organoids of MDA-MB-231 xenografts ***P < 0.001, **P < 0.01, *P < 0.05 were considered statistically significant
Fig. 2
Fig. 2
Next-generation RNA sequencing and RT-qPCR analysis in MDA-MB-231 cells. A Heat map image of RNA sequencing data (n = 3), where “n” represents the number of biological replicates per group. B Volcano plot showing that CBD (2.5 μM) treatment induced upregulated (in orange) and downregulated (in blue) genes. C Bar graphs showing KEGG pathway analysis of CBD (2.5 μM) upregulated genes. D Bar graphs showing KEGG pathway analysis of CBD (2.5 μM) downregulated genes. E Illustrative images of CBD-induced up- (GADD45A, GADD45G, and CCNB2) and downregulated (LOXL4, FASN, and ITGB4) genes. F RT-qPCR analysis of GADD45A, GADD45G, LOX, FASN, and CCND1 genes. Control group represents untreated MDA-MB-231 cells. ***P < 0.001, **P < 0.01, *P < 0.05 were considered to be statistically significant
Fig. 3
Fig. 3
Effect of CBD on GADD45-alpha, p-p38, and p53 proteins in TNBC cells. A Immunoblots and densitometry data of GADD45-alpha, p-p38, and p53 proteins upon CBD treatment in MDA-MB-231 cells. B Immunoblots and densitometry data of GADD45-alpha, p-p38, and p53 proteins upon CBD treatment in MDA-MB-468 cells. After ascertaining p-p38 expression, we performed stripping and reprobed the same blot with β-actin antibody. C Immunostaining with GADD45α antibody. ***P < 0.001, **P < 0.01, *P < 0.05 were considered to be statistically significant
Fig. 4
Fig. 4
Effect of CBD on integrin-α5, integrin-β5, and integrin-β1 proteins in TNBC cells. A, B Immunoblots and densitometry data of integrin-α5, integrin-β5, and integrin-β1 proteins in TNBC cells. After ascertaining the Integrin β1 expression, we performed stripping and reprobed the same blot with Integrin α5 and Integrin β5 antibodies. C Immunoblots and densitometry data of integrin-α5, -β5, and -β1 proteins in MDA-MB-468 cells (3D). After investigating the Integrin β5 expression, we performed stripping and the same blot was reprobed with Integrin β1 antibody. ***P < 0.001, **P < 0.01, *P < 0.05 were considered to be statistically significant
Fig. 5
Fig. 5
CBD decreased the migration of TNBC cells. A xCELLigence CIM-16 plate migration-based assay in MDA-MB-231 cells exposed to CBD. B, C Immunoblots and densitometry data of fibronectin and vimentin proteins in TNBC cells. ***P < 0.001, **P < 0.01, *P < 0.05 were considered to be statistically significant
Fig. 6
Fig. 6
Chemo-sensitization effects of CBD in TNBC cells. A, B Anti-cancer activity of Synthetic CBD (1 μM and 2.5 μM) and DOX combination in 2D cultures of TNBC cells. C, D Anti-cancer activity of Synthetic CBD (5 μM) and DOX combination in 3D cultures of TNBC cells. E xCelligence CIM-16 plate migration-based assay in MDA-MB-231 cells with CBD and DOX combination in MDA-MB-231 cells. F Immunoblots and densitometry data of caspase 9, LOX, and integrin-α5 proteins in CBD pre-treated MDA-MB-468 cells (24 h)and further treated with DOX treatment (2 days). After ascertaining LOX expression, we performed stripping and reprobed the same blot with caspase 9 and β-actin antibodies. Statistical significance considerations: ***P < 0.001, **P < 0.01, and *P < 0.05 vs control; ##P < 0.01 significant vs DOX (1 μM); $ $ $P < 0.001, $P < 0.05 vs CBD (1 μM)
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