Honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and -independent apoptosis

Abstract

Honokiol (HNK) is an active component purified from magnolia, a plant used in traditional Chinese and Japanese medicine. Here we show that HNK significantly induces cytotoxicity in human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM. Neither coculture with bone marrow stromal cells nor cytokines (interleukin-6 and insulin-like growth factor-1) protect against HNK-induced cytotoxicity. Although activation of caspases 3, 7, 8, and 9 is triggered by HNK, the pan-caspase inhibitor z-VAD-fmk does not abrogate HNK-induced apoptosis. Importantly, release of an executioner of caspase-independent apoptosis, apoptosis-inducing factor (AIF), from mitochondria is induced by HNK treatment. HNK induces apoptosis in the SU-DHL4 cell line, which has low levels of caspase 3 and 8 associated with resistance to both conventional and novel drugs. These results suggest that HNK induces apoptosis via both caspase-dependent and -independent pathways. Furthermore, HNK enhances MM cell cytotoxicity and apoptosis induced by bortezomib. In addition to its direct cytotoxicity to MM cells, HNK also represses tube formation by endothelial cells, suggesting that HNK inhibits neovascurization in the bone marrow microenvironment. Taken together, our results provide the preclinical rationale for clinical protocols of HNK to improve patient outcome in MM.

Figure 1.

HNK induces cytotoxicity in MM cell lines and tumor cells from patients with MM, but not in normal PBMNCs. (A, B) Growth inhibition in MM cell lines by HNK was assessed by colorimetric assay after 48-hour culture. Data represent mean plus or minus the standard deviation (SD) of 3 independent experiments (^*P < .05 versus untreated cells). (C) Viability of PBMNCs derived from 7 healthy subjects was assessed by colorimetric assay after 48-hour culture. Data represent mean plus or minus SD of triplicate cultures (^*P < .05 versus untreated cells). (D) Cytotoxicity of HNK against patient MM cells was determined by comparison of percentage of CD38^high cells after culture with HNK (4, 6, 8, and 10 μg/mL) versus media for 48 hours. HNK induced cytotoxicity to patient MM cells in a dose-dependent manner (n = 6, values represent the mean plus or minus SD, ^*P < .005 versus untreated cells). Double asterisks indicate statistical significance in 2 lines; triple asterisks, in 3 lines.

Figure 2.

HNK induces apoptosis in MM cells. (A) MM.1S and RPMI 8226 cells were treated with 8 μg/mL HNK for 48 hours, and apoptosis was assessed using TUNEL assay. (B) Cleavage of caspases and PARP was determined by Western blotting of MM.1S whole-cell lysates after 10 μg/mL HNK treatment for 12 hours and 24 hours, with or without z-VAD-fmk (25 μM) preincubation for 1.5 hours. (C) MM.1S cells were treated with HNK or As₂O₃, with or without 25 μM z-VAD-fmk pretreatment for 1.5 hours. Activation of caspase 3 was determined by flow cytometry. (D) MM cells were treated with HNK or As₂O₃ for 24 hours, with or without z-VAD-fmk (25 μM, 100 μM) pretreatment for 1.5 hours. Induction of apoptosis and cytotoxicity was determined by flow cytometry after APO2.7 staining and trypan blue exclusion, respectively. Values represent the mean plus or minus SD for 3 independent experiments. (E) MM.1S cells were treated with HNK (10 μg/mL for 0, 4, 8 and 12 hours). Whole-cell lysates were subjected to Western blotting to assess the expression of Bcl-2 family proteins. (F) MM.1S cells were treated with HNK (10 μg/mL for 24 hours), with or without pretreatment by z-VAD-fmk. Proteins in cytosolic fraction were subjected to immunoblotting of AIF and Endo G.

Figure 3.

Combination of HNK with bortezomib enhances cytotoxicity against MM.1S cells. (A) MM.1S cells were treated with HNK and bortezomib for 48 hours and cell growth was determined by colorimetric assay. Values represent the mean plus or minus SD of triplicate cultures (^*P < .05). (B) MM.1S cells were treated with HNK and bortezomib for 48 hours and induction of apoptosis was determined by APO2.7. Values represent the mean plus or minus SD of 2 independent cultures (^*P < .05). (C) MM.1S cells were treated with HNK and bortezomib for 8 hours. Whole-cell lysates were subjected to Western blotting to assess phosphorylation and protein expression of Hsp27, Hsp70, and Mcl-1.

Figure 4.

HNK overcomes the protective effects of IL-6, IGF-1, and adherence to patient BMSCs. MM.1S cells were treated for 48 hours with indicated concentrations of HNK in the presence or absence of IL-6 (A), IGF (B), or BMSCs derived from 2 patients with MM (C, D). DNA synthesis was determined by measuring [³H]-thymidine incorporation during the last 8 hours of 48-hour cultures. Values represent the mean plus or minus SD of triplicate cultures (^*P < .05, ^**P < .05 inversely significant).

Figure 5.

HNK modulates growth and survival signaling pathways in MM.1S cells. (A) MM.1S cells were pretreated with HNK (10 μg/mL) in 2.5% FCS containing media for 3 hours and 6 hours; cells were then stimulated with IL-6 (10 ng/mL) for 10 and 20 minutes. Whole-cell lysates were subjected to Western blotting to assess phosphorylation and protein expression of STAT3, ERK2, and Akt. (B) MM.1S cells were pretreated with HNK (10 μg/mL) in 2.5% FCS containing media for 3 hours and 6 hours, and then stimulated with IGF-1 (25 ng/mL) for 10 and 20 minutes. Whole-cell lysates were subjected to Western blotting for phosphorylation and protein expression of ERK2 and Akt. (C) MM.1S cells were pretreated with HNK (10 μg/mL) in 2.5% FCS containing media for 3 hours and 6 hours. Whole-cell lysates were subjected to Western blotting to determine cleavage of caspases and expression of gp80 and gp130.

Figure 6.

HNK significantly inhibited angiogenesis of HUVECs. HUVECs were cultured with (B) or without (A) 8 μg/mL HNK for 6 hours, and tube formation was assessed. Original magnification ×40.