Pro-Apoptotic Activity of Honokiol Analogues in B-Cell Lymphoid Malignancies

Honokiol, a small-molecule polyphenol isolated from Magnolia officinalis, was recently found to have antitumor properties. The agent showed cytotoxicity against CLL cells, but so far there are no data on its activity in other lymphoid tumors. More recently, new honokiol analogues (HAs) were synthetized, however, their cytotoxicity has not been investigated in leukemia/lymphoma yet. The aim: This is the first study assessing pro-apoptotic activity of new HAs on tumor cells from B-cell lymphoid malignancies. Material and Methods: Seven HAs: BR MMK + phloroG-3,3' dibromo4,4' bis (dimethlyamino) benzylidenephloroglucinol (HA 1), dibromoimipramine blue (HA 2), 3 bromo-4 dimethylamino diuhydroxyphenoxane (HA 3), bromo Gentian Violet-tribromogentian violet (HA 4), Gentian Violet (HA 5), BR dimethylaminobenzaldehyde + phloroG 3 bromo-4 dimethlyaminobenzylidenephloroglucinol (HA 6) and hexafluoro-diallylhexaflurobisphenol (HA 7) were studied. We assed ex vivo CLL cells (21 pts) and, in in vitro settings, pre-B-cell acute lymphoblastic leukemia (NALM-6), Burkitt lymphoma (Raji), diffuse large B-cell lymphoma; DLBCL (Toledo) and multiple myeloma (MM)-derived (RPMI 8226) cell lines. Lymphocytes obtained from 10 healthy volunteers were also treated. HAs were tested in concentrations 1–10 mM (24 and 48 hr incubation), then the minimal doses that triggered significant apoptosis and 48 hr time point were chosen for further experiments. Overall cytotoxicity of HAs was estimated using propydium iodide (PI) flow cytometry assay. The half maximal inhibitory concentration (IC₅₀) of the study-drugs was estimated. Drug-induced apoptosis was assessed by the Annexin-V assay. Compensated apoptotic index (CAI) was calculated as a difference in percent of Annexin-V-positive cells between the drug-treated sample and the parallel untreated culture. Activation of caspases-8, -9 and -3 as well as expression of several apoptosis–regulating proteins, including Bcl-2 family (Bax, Bak, Bcl-2, Mcl-1), and inhibitor of apoptosis protein (IAP) family (cIAP1, cIAP2, XIAP, Smac/DIABLO) and IAP antagonists (survivin, HTRA2/Omi) were also investigated. Results: HA 1 triggered significant apoptosis in CLL cells starting from the dose 5mM, with minimal significant CAI (msCAI) 12.5%; vs. control - p=0.043 (IC₅₀ 10mM). In Raji cells msCAI was 17% at the dose 0.5mM; p=0.025 (IC₅₀ 2.5mM). In Toledo msCAI was 28% (at 0.1mM); p=0.007 (IC50 0.5mM) and RPMI 8226 (msCAI 58% at 0.5mM; p<0.001, IC₅₀ 0.5mM) cells. HA 2 induced both msCAI and IC50 at 2.5mM (msCAI 51.9%; p<0.001) In Raji cells msCAI was 22.1% (at 1mM); p=0.025, with IC₅₀ 2.5mM. The highest anti-tumor effect of HA 2 was found for Toledo (msCAI 52% at 0.5mM; p<0.001, IC₅₀ 0.5mM) and RPMI 8226 (msCAI 45.7% at 0.1mM; p<0.001, IC₅₀ 0.5mM) cell lines. HA 4 in CLL cells msCAI induced 15,8% (0.1mM); p=0.0270, IC50 10mM). In Raji cells msCAI was 27.4% (at 2.5mM); p=0.015 (IC₅₀ 5mM). Toledo and RPMI 8226 showed msCAI and IC₅₀ at the same dose 2.5mM; msCAIs were 65.2% and 67.5%, respectively; p<0.0001. HA 5 in CLL cells triggered msCAI 23% (p=0.012) at 2.5mM (IC₅₀ 0.5mM). In Raji model msCAI was 78.5 % at 0.1mM; p<0.001 (IC₅₀ 0.1mM). In Toledo msCAI was 65% (at 0.5mM); p<0.001 (IC₅₀ 0.5mM). Similarly, in RPMI 8226 cells msCAI was 59.3% (at 0.5mM); p<0.001 (IC₅₀ 0.5mM). Interestingly, HAs did not trigger apoptosis of healthy lymphocytes at doses inducing msCAI. Their mechanism of action of HAs 1,2 4 and 5 was caspase-dependent apoptosis, triggered through both TNF receptor and mitochondrial pathways. In CLL cells their significantly downregulated Bcl-2, Mcl-1, cIAP-1, XIAP and Smac/DIABLO proteins and upregulated survivin. In Raji cells, significant Bcl-2 downregulation, with upregulation of Bax and Bak proteins was found. In Toledo cells decreased expression Bcl-2 and XIAP and overexpression of Bax and survivin were observed. In RPMI 8226 cells, HAs significantly downregulated Bcl-2 and XIAP and upregulated Bax, Bak and survivin proteins. In contrast, NALM-60 cells were resistant to all HAs. Moreover, HA 3, 6 and 7 did not significantly affect the examined malignant cells. Conclusions: These data indicate that HAs some are potent tumor-selective inducers of apoptosis in ex vivo CLL cells and in in vitro model of Burkitt lymphoma, DLBCL and MM. Those HAs should be examined for further clinical application either as a single agents or in combination with other anti-cancer drugs.

No relevant conflicts of interest to declare.