Cancer Chemotherapy and Pharmacology https://doi.org/10.1007/s00280-018-3547-2

ORIGINAL ARTICLE

Antitumor activity of the microtubule inhibitor MBRI-001 against human hepatocellular carcinoma as monotherapy or in combination with sorafenib Mengyan Deng1   · Linna Li3 · Jianchun Zhao1,4 · Shoujun Yuan3 · Wenbao Li1,2,4 Received: 9 December 2017 / Accepted: 20 February 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Purpose  MBRI-001 is a novel synthetic derivative of plinabulin. In this study, our purpose is to investigate the inhibition effects of MBRI-001 on human hepatocellular carcinoma as monotherapy or in combination with sorafenib. Methods  HCCLM3 and Bel-7402 cell lines were used for activity evaluation in vitro. The anti-proliferative activity of MBRI-001 was assessed by MTT assay. The morphological change of microtubules was determined by immunofluorescence assay. The cell cycle was measured by flow cytometer. The expression of cyclin B1 (CCNB1) was analyzed by RT-qPCR and western blotting assays. The antitumor activities in vivo were evaluated with human HCC xenograft mice model. Results  Our data demonstrated that MBRI-001 had better anti-proliferative activities than that of plinabulin against HCCLM3 and Bel-7402 cell lines. MBRI-001 inhibited the formation of microtubules and induced G2/M arrest with the downregulation of CCNB1. In vivo orthotopic mice model demonstrated that MBRI-001 significantly inhibited the growth of HCCLM3 with the apoptosis and necrosis observed in tumor. The combination treatment of MBRI-001 with sorafenib in subcutaneous mice model exhibited a higher antitumor inhibition rate at 72.0%, in comparison with MBRI-001 or sorafenib as monotherapy at 40.7% or 47.7%, respectively. Conclusion  MBRI-001 had better inhibition effects on microtubules and human hepatocellular carcinoma than that of plinabulin. The combination treatment of MBRI-001 and sorafenib exhibited a higher antitumor effect, which could provide a new strategy to treat HCC in the future. Keywords  MBRI-001 · Hepatocellular carcinoma (HCC) · Microtubules inhibitor · Sorafenib · Combination treatment

Introduction

* Shoujun Yuan [email protected] * Wenbao Li [email protected] 1



School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China

2



Innovation Center for Marine Drug Screening and Evaluation, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China

3

Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China

4

Marine Biomedical Research Institute of Qingdao, Qingdao 266071, China



Polymerized microtubules are major components of mitotic spindles that play an important role in the separation of homologous chromosomes [1, 2]. Inhibiting the polymerization and formation of microtubules can induce irregular chromosome alignment and eventually lead to apoptosis of cancer cells [3, 4]. Consequently, microtubules have become attractive targets for the development of anticancer drugs, such as paclitaxel and vincristine [5, 6]. Plinabulin, a synthetic analog of the marine natural product “diketopiperazine phenylahistin” (Fig. 1), showed potent depolymerization effects on microtubules [7, 8]. Currently, the combination therapy of plinabulin and docetaxel has been pushed into Clinical Phase III trials [9, 10]. To obtain compounds possessing lower toxicities and better antitumor effects, our laboratory has designed and synthesized a series of deuteriumsubstituted derivatives of plinabulin. Our previous research

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Fig. 1  Structures of plinabulin and MBRI-001

showed that MBRI-001 (Fig. 1), one of these derivatives, could significantly inhibit the growth of NCI-H460 cell line, and MBRI-001 had better pharmacokinetic properties than that of plinabulin, such as a longer half-life and a higher area under curve [11]. However, the antitumor inhibition rate was still very low when MBRI-001 was administrated alone against NCI-H460 xenograft tumor model in nude mice [11]. In addition, the action mechanism of MBRI-001 is not clear and needs to be further investigated. Therefore, we continue to evaluate the antitumor activity and the action mechanism of MBRI-001 in this study. Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world, and it ranks as the third leading cause of cancer-related death [12, 13]. However, few studies are reported on the application of microtubule inhibitors in HCC. Sorafenib is a FDA-approved drug for the treatment of metastatic and advanced HCC [14, 15]. However, after several months of treatment, numerous HCC patients were found either with poor response or even with developed resistance [16]. To enhance the efficacy and overcome the limitations observed in clinic, a number of combination studies with sorafenib have been reported [17–19]. In the present study, the anti-proliferative activity of MBRI-001 was evaluated against HCCLM3 and Bel-7402 cell lines. The action mechanism of MBRI-001 was preliminarily explored through the detection of microtubule formation, the cell cycle distribution and the expression of cyclin B1 (CCNB1). The antitumor activity of MBRI-001 in vivo was assessed using an orthotopic mice model. Additionally, the combination effects of MBRI-001 and sorafenib on HCCLM3 cell line were determined with a subcutaneous mice model in vivo.

Cancer Chemotherapy and Pharmacology

diluted to proper concentration with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY). The terminal concentration of DMSO was < 0.1%. Human HCC cell lines HCCLM3, HCCLM3-luc-GFP and Bel-7402 were gifted from Beijing Institute of Radiation Medicine. These cell lines were cultured in DMEM (Gibco, Grand Island, NY) medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (YuanHeng ShengMa, Beijing, China) at 37 °C in 5% ­CO2.

Cell viability assay HCCLM3 and Bel-7402 cells were seeded at 3.0–5.0 × 103 cells/well in 96-well flat-bottomed plates and incubated overnight at 37 °C. Both cell lines were treated with MBRI-001 or plinabulin (0–100 nM) for 72 h. The inhibition rates were evaluated by MTT (JKHD, Beijing, China) assay. Briefly, the medium was replaced by 100 µL of fresh medium containing 0.5 mg/mL MTT per well. After incubation at 37 °C for 4 h, the medium was removed, then 200 µL of DMSO was added into each well. The absorbance of each well was measured at 570 nm on a Microplate Reader (Thermo Fisher Scientific, Rochester, NY, USA). Dose–response curves of triplicate experiments were analyzed via Origin 8.

Immunofluorescence assay

Materials

2.0–5.0 × 105 HCCLM3 cells/well were seeded in 6-well chamber slides and treated with MBRI-001 or plinabulin (12.5 nM) for 24 h. The cells were washed three times with PBS and fixed with 4% cold paraformaldehyde at 4 °C for 15 min. The cells were permeabilized in 0.05% Triton X-100 for 10 min, and then blocked in 1% BSA for 30 min. Immediately, the cells were incubated with primary antibody (β-tubulin [1:100], Servicebio, Wuhan, China) at 4 °C overnight, followed by incubation with secondary antibody (anti–mouse CY3 [1:300], servicebio, Wuhan, China) for 1 h. The cells were mounted with anti-fade mounting medium (Servicebio, Wuhan, China) and observed using a fluorescence microscope (ECLIPSE C1, Nikon). The nuclei was stained with 4, 6-diamidino-2-phenylindole (DAPI) (Servicebio, Wuhan, China). The immunofluorescence optical density (IOD) of β-tubulin (red fluorescence) was measured using Image-pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Reagents and cell lines

Cell cycle assay

MBRI-001 (purity: 99.9%) was synthesized according to the published reaction routes [11]. Sorafenib was purchased from Nanjing First Pharmaceutical Technology CO., LTD (Nanjing, China). These compounds were dissolved in dimethylsulfoxide (DMSO) (AMRESCO, Solon, OH) and

To analyze the effects of MBRI-001 or plinabulin on cell cycle distribution, HCCLM3 and Bel-7402 cells were seeded in 6-well flat-bottom plates (2.0–5.0 × 105 cells/well) and treated with MBRI-001 or plinabulin (0, 12.5, 25, 50 nM) for 24 h. The collected cells from the control or the drug-treated

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Cancer Chemotherapy and Pharmacology

groups were fixed with 50% cold ethanol at 4 °C for 45 min, incubated with 0.1 mg/mL RNase for 30 min and stained with 50 µg/mL of PI (Solarbio, Beijing, China) for 5 min. Then, the distribution of cell cycle was analyzed on a flow cytometer (Millipore, Billerica, MA, USA). The cell cycle distribution were analyzed and then quantified using ModFit LT (Verity Software House, USA).

RT‑qPCR assay HCCLM3 and Bel-7402 cells were seeded in 6-well flat-bottom plates (2.0–5.0 × 105 cells/well) and treated with MBRI001 or plinabulin (0, 12.5, 50 nM) for 24 h. Then total RNA was extracted from the control and the drug-treated groups using a TRIzon kit (CWBIO, Taizhou, China). The firststrand synthesis of cDNA was carried out with HiFiScript cDNA Synthesis Kit (CWBIO, Taizhou, China) following the manufacturer’s instructions. RT-qPCR reactions were performed using UltraSYBR Mixture (CWBIO, China) with primers of β-Actin and CCNB1 (Invitrogen, Carlsbad, CA): β-Actin F 5′-TTC​CTT​CCT​GGG​CAT​GGA​GTC​CTG​-3′; R 5′-GAG​GAG​CAA​TGA​TCT​TTG​ATC​TTC​A-3′. CCNB1 F 5′-TTG​GGG​ACA​TTG​G-TAA​CAA​AGTC-3′; R 5′-ATA​GGC​TCA​GGC​GAA​AGT​TTTT-3′.

Western blotting assay HCCLM3 and Bel-7402 cells were treated with MBRI-001 or plinabulin (0, 12.5, 50 nM) for 24 h. To obtain protein lysates, cells collected from the control or the drug-treated groups were suspended in lysis buffer. Proteins were then quantitated using Bradford Protein Assay Kit (CWBIO, Taizhou, China). Subsequently, equal amounts of protein were loaded (60 µg/lane) onto a 10% SDS–PAGE Gel. Electrophoresis was performed at 150 V for 60 min to make proteins separated. Then, the proteins were transferred onto a nitrocellulose membrane at 60 V for 60 min. After blocked with 5% non-fat milk for 1 h, the membrane was incubated with primary antibodies against β-Actin (CWBIO, Taizhou, China) and CCNB1 (ABclonal, Wuhan, China). The membrane was then incubated with the HRP-conjugated secondary antibody (1:10,000) for 1 h and visualized using the enhanced chemiluminescence (Thermo Fisher Scientific, Rochester, NY, USA) detection system.

Human hepatocellular carcinoma orthotopic mice model To evaluate the antitumor effect of MBRI-001 in vivo, the bioluminescence imaging (BLI) technique was applied in human HCC orthotropic mice model. Briefly, 2.0 × 106 HCCLM3-luc-GFP cells in 200  µL of PBS were orthotopically inoculated into the sub-serosa layer of liver wall

in female athymic nude mice (n = 12) (Vital River Labs, Beijing, China). Two weeks after surgery, the mice were observed by bioluminescence imager (IVIS Spectrum CT, Etaluma, CA, USA) after the intraperitoneal injection of 150 mg/kg of Luciferin. According to the total flux [p/s] emitted by HCCLM3-luc-GFP cells, the mice were randomly divided into three groups receiving 6 mg/kg MBRI001 intravenously once 2 days, 12.5 mg/kg docetaxel intravenously once 2 days or control vehicle for 21 days. The solution for MBRI-001 consisted of 8% propylene glycol, 12% HS-15 and 80% physiological saline. The tumor growth was assessed once weekly by measuring the total flux [p/s]. The mice body weight was monitored once 2 days. The tumor weight was measured at the end of experiment. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

In situ detection of apoptosis Tumor tissues of the control group or MBRI-001-treated group were analyzed using hematoxylin-eosin (H&E) staining and immunohistochemical (IHC) staining with the primary antibody against cleavage of caspase-3 as previously described [20]. In addition, apoptosis of HCCLM3 was evaluated using a TdT-mediated dUTP Nick-End Labeling (TUNEL) Apoptosis Assay Kit (Servicebio, Wuhan, China) in accordance with the manufacturer’s instructions.

Combination treatment of MBRI‑001 and sorafenib To assess the interaction between MBRI-001 and sorafenib in vitro, HCCLM3 cells were seeded in 96-well plates at 3.0–5.0 × 103 cells/well and exposed to single-agent doses and combination doses for 72 h. In single-agent doses, concentrations of MBRI-001 and sorafenib were ranged from 1.5625 to 100 nM and 1.5625 to 100 µM, respectively. In combination doses, sorafenib was set at 6.25 µM exhibiting 20–30% inhibition rate, to combine with different concentrations of MBRI-001 ranged from 3.125 to 50 nM. The inhibition rate was tested using MTT assay. The combination activity of MBRI-001 and sorafenib in vivo was examined using the HCC subcutaneous mice model. Briefly, 5.0 × 10 6 HCCLM3 cells in 200  µL of PBS were subcutaneously injected into the right flank of female athymic nude mice (n = 20; Vital River Labs, China). When the tumor volume reached 100–150 mm 3, the mice were randomly divided into four groups receiving 30 mg/kg sorafenib orally once daily, 6 mg/kg MBRI-001 intravenously once 2 days, the combination agents or the control vehicle for 14 days. Sorafenib was suspended in 1% carboxymethylcellulose sodium (CMC-Na) at 3 mg/ mL. MBRI-001 was dissolved in the solution consisted of 8% propylene glycol, 12% HS-15 and 80% physiological

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saline at 0.6 mg/mL. The tumor volume and mice weight were measured once every 3 days. Tumor volume (TV) was calculated as TV (­ mm3) = length (L) × width (W)2/2. The tumor weight was monitored at the end of experiment. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Statistical analysis GraphPad Prism 5 was used for statistical analysis. Significance of statistical differences in the drug-treated groups versus the control group was determined using the Student’s t test. P < 0.05 was considered as the minimal level of statistical significance.

Results Anti‑HCC effects of MBRI‑001 and plinabulin The anti-proliferative effects of MBRI-001 and plinabulin against HCCLM3 and Bel-7402 cell lines were evaluated by MTT assay. The results indicated that MBRI-001 had better anti-HCC activities than that of plinabulin (Fig. 2a). The ­IC50 values of MBRI-001 were 5.58 nM against HCCLM3 cell line and 5.70 nM against Bel-7402 cell line, whereas the ­IC50 values of plinabulin were 7.24 nM and 11.73 nM, respectively. Effects of MBRI-001 and plinabulin on the formation of microtubules were examined by immunofluorescence technique. Figure 2b indicated that the β-tubulin stained as red fluorescence was changed from normal and regular filamentous to irregular and shrinking form by MBRI-001 or plinabulin at 12.5 nM. Fluorescence images (Fig. 2c) showed that IOD values of β-tubulin (shown as red fluorescence) in the control, MBRI-001-treated or plinabulin-treated groups were 7027, 5630 or 3997, respectively. These results indicated that MBRI-001 had better inhibition activity than that of plinabulin in the formation of microtubules. Cell cycle was determined by flow cytometer after the treatment of MBRI-001 or plinabulin (0, 12.5, 25, 50 nM) for 24 h. The results demonstrated that percentage of cells in G2/M period increased with the concentration gradient of MBRI-001 or plinabulin (Fig. 2d). In addition, MBRI-001 at 12.5 nM could induce 34–42% HCC cells accumulated in G2/M period, whereas plinabulin at the same concentration only could induce 12% (Fig. 2d). These results indicated that MBRI-001 had better cell cycle arrest ability in comparison with plinabulin, which was consistent with the results of immunofluorescence shown in Fig. 2c.

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Cancer Chemotherapy and Pharmacology

RT‑qPCR and western blotting assays The expression of CCNB1 was investigated in HCCLM3 and Bel-7402 cell lines using RT-qPCR and western blotting assays. As shown in Fig. 3, RT-qPCR (Fig. 3a) and western blotting assays (Fig. 3b) indicated that the mRNA and protein levels of CCNB1 were downregulated by MBRI-001 or plinabulin in a dose-dependent manner. More significant downregulation was observed in MBRI-001-treated group in comparison with plinabulin-treated group.

Apoptosis and necrosis induced by MBRI‑001 in human hepatocellular carcinoma orthotopic mice model Having shown the superior anti-HCC viability of MBRI-001 in vitro, the efficacy of MBRI-001 in vivo was investigated using HCC orthotropic mice model. The total flux [p/s] emitted by HCCLM3-luc-GFP cells in the liver of mice was detected by bioluminescence imager. During the experimental period, the total flux [p/s] in MBRI-001-treated group and docetaxel-treated group decreased gradually in a timedependent manner (Fig. 4a), which indicated the inhibition effect of MBRI-001 and docetaxel on the growth of HCCLM3-luc-GFP cells in orthotopic tumor. The inhibition rate of docetaxel was 91.4%, but it induced significant body weight loss, which indicated the severe side effects. After the treatment with 6 mg/kg MBRI-001 for 21 days, the total flux [p/s] decreased to approximately 1.5 magnitude orders compared with that in the control group (Fig. 4a). The calculated inhibition rate of MBRI-001 was 91.7%, which suggested that MBRI-001 had a very strong antitumor activity. In addition, there was no significant change in mice body weight during the treatment (Fig. 4b). However, the calculated inhibition rate of MBRI-001 was only 32.1% according to the tumor weight monitored at the end of experiment (Fig. 4c). To explain the difference of above calculated inhibition rates obtained from two different assays, the tumor tissues from the control group or MBRI-001-treated group were evaluated with H&E staining, IHC staining and TUNEL assay. As shown in Fig. 5, the condensed and fragmented nuclei were observed in H&E staining, which revealed the apoptotic lesions from tumor tissues. In comparison, the apoptotic region in MBRI-001-treated group were more significant than that in control group. In comparison with the control group, more massive cleavage of caspase-3 was induced by MBRI-001-treated group in IHC-staining assay (Fig. 5). In addition, the results of TUNEL assay demonstrated that the quantity of the apoptotic cells stayed at late stage in MBRI-001-treated group was more than that in control group. Taken together, these data demonstrated that MBRI-001 could inhibit the growth of HCCLM3 cell lines in vivo with significant apoptosis and necrosis observed in

Cancer Chemotherapy and Pharmacology Fig. 2  MBRI-001 and plinabulin inhibited the proliferation of HCC and triggered G2/M arrest. a Cell viabilities of HCCLM3 and Bel-7402 cell lines were measured by MTT assay after treatment of MBRI001 or plinabulin (dose range: 1.56–100 nM) for 72 h. Data presented were mean ± SD of three independent experiments. b HCCLM3 cells were treated with MBRI-001 or plinabulin (12.5 nM) for 24 h and then stained for β-tubulin and DAPI. Figures presented were representative of 2 independent experiments. c IOD values of β-tubulin (red fluorescence) in control, MBRI-001-treated and plinabulin-treated groups. Error bars indicated SEM of two independent experiments. d Cell cycle distribution of HCCLM3 or Bel-7402 cell lines was measured with flow cytometer after the treatment of MBRI001 or plinabulin (0, 12.5, 25, 50 nM) for 24 h. Figures were representative of two independent experiments

tumor tissues, although the tumor weight did not change significantly at the end of experiment.

Combination treatment of MBRI‑001 and sorafenib Combination studies of MBRI-001 and sorafenib were conducted in HCCLM3 cell line. In vitro results indicated that

single-agent doses and combination doses inhibited cell proliferation in a dose-dependent manner (Fig. 6a). As shown in Fig. 6a, the inhibition rates of combination were significantly higher than that of monotherapy, which suggested the favorable effects between MBRI-001 and sorafenib. The HCCLM3 subcutaneous mice model was conducted to evaluate the combination efficacy of MBRI-001

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Cancer Chemotherapy and Pharmacology

Fig. 3  MBRI-001-induced G2/M period arrest was associated with the downregulation of CCNB1 (M1 and M2 represented 12.5 and 50  nM of MBRI-001. P1 and P2 represented 12.5 and 50  nM of plinabulin. C represented control). a Total RNA from HCC cells was assessed using RT-qPCR method after treatment of MBRI-001 or

plinabulin (12.5 and 50  nM) for 24  h. b Total protein lysates from above groups were analyzed by western blot analysis with the antibody against CCNB1. Data were the representation of two independent experiments

and sorafenib in vivo. According to the measurement of tumor volume (Fig. 6b), the inhibition rate of the combination group was 71.8%, which was much higher than that in MBRI-001-treated group (41.2%) or sorafenib-treated group (47.1%). Similar results were obtained based on the measurement of tumor weight at the end of experiment. The inhibition rates of the combination-treated group, MBRI-001treated group or sorafenib-treated group were 71.8%, 40.7%, or 47.1%, respectively (Fig. 6c). These results indicated that the combination treatment of MBRI-001 and sorafenib could significantly enhance the inhibition effect of monotherapy on the growth of HCCLM3 cells in vivo. In addition, the combination treatment did not result in any obvious weight loss (Fig. 6d).

­(IC50: 7–12 nM). The IOD value of β-tubulin in MBRI-001treated group (dose at 12.5 nM) was about 1000 lower than that in plinabulin-treated group (dose at 12.5 nM). Some reports have showed that the carbon–deuterium bond is more stable than the carbon–hydrogen bond, which may play a major role in improving PK properties and efficacy [11, 24]. The better antitumor activities of MBRI-001 may be due, at least in part, to the carbon–deuterium bond. Microtubule-targeting agents can affect the microtubule cytoskeleton and prevent the normal division course of cells, which eventually cause mitotic arrest and cell death by activating the checkpoint protein in mitosis [25, 26]. CCNB1, a regulatory protein, plays an important role in pushing the cell cycle from G2 to M phase. The downregulation of CCNB1 has been observed in G2/M arrest [27, 28]. In the present study, HCCLM3 and Bel-7402 cell lines were blocked in G2/M phase by MBRI-001. The cells blocked in G2/M phase by MBRI-001 were 22–30% more than that by plinabulin at 12.5 nM, which is consistent with immunofluorescence results. Analysis of mRNA and protein expression levels of CCNB1 demonstrated that MBRI-001 induced more significant downregulation of CCNB1 than that of plinabulin at either 12.5 nM or 50 nM (Fig. 3). The in vivo orthotopic mice model demonstrated that MBRI-001 could significantly inhibit the tumor growth with apoptosis and necrosis observed in tumor tissues. However, it was still needed to find a way to enhance the low inhibition rate of MBRI-001 as monotherapy observed in tumor weight. Currently, sorafenib is the only approved first-line targeted drug against advanced hepatocellular carcinoma (HCC). However, acquired resistance and undesirable side effects of sorafenib imply the urgent need to develop more effective combination strategies for treating HCC [16–18]. Additionally, the acquired resistance of

Discussion Microtubule-targeting agents could be classified into at least three primary categories according to their binding sites on tubulin, namely as colchicine-, vinca alkaloid- and paclitaxel-binding sites [21, 22]. Vinblastine that targets the vinca alkaloid site and paclitaxel that targets the paclitaxel site have been developed as the first-line drugs for cancer therapy. However, the colchicine-binding site inhibitors, with the longest history of research, have not been approved because of the significant toxicity on nerve cells and bone marrow [23]. MBRI-001 is a deuterated derivative of plinabulin, which has been reported targeting the colchicine-binding site [10, 11]. In this study, we found that MBRI-001 could significantly reduce the viability of hepatocellular carcinoma cells and inhibit the formation of microtubules. Additionally, our data indicated that MBRI-001 had better anti-HCC activities ­(IC50: 5–6 nM) than that of plinabulin

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Cancer Chemotherapy and Pharmacology Fig. 4  Anti-HCC activity of MBRI-001 in vivo. a Mice were treated intraperitoneally with MBRI-001 (6 mg/kg), docetaxel (12.5 mg/kg) or vehicle alone once two days for 3 weeks. The value of total flux [p/s] was measured once a week. *P < 0.05, **P < 0.01 and ***P < 0.001. b Body weight of mice was monitored once 2 days. Data were expressed as mean ± SEM of six mice in each group. c Tumor weight was monitored at the end of experiment

almost a single chemotherapy drug has been observed in the treatment of hepatocellular carcinoma [29, 30]. Combination therapies with different targets are prevalent in cancer treatment. Therefore, the combination study of MBRI-001 and sorafenib was performed in this study. In vitro curves

shown in Fig. 6a suggested the favorable effects of MBRI001 and sorafenib combination. In vivo subcutaneous mice model indicated that the combination treatment could improve the inhibition rate greatly compared with MBRI001 or sorafenib as monotherapy; while the inhibition rate

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Cancer Chemotherapy and Pharmacology

of combination treatment was 72.0%, in comparison with the inhibition rates of MBRI-001 or sorafenib monotherapy was 40.7% or 47.7%, respectively. In conclusion, our data demonstrated that MBRI001, a novel synthetic analog of plinabulin, could inhibit the formation of microtubules and induce G2/M phase arrest through downregulating the key regulatory protein CCNB1. In addition, MBRI-001 could inhibit the growth of HCCLM3 in orthotopic mice model with both apoptosis and necrosis observed in tumor tissue. The combination study revealed that the combination treatment of MBRI001 and sorafenib could be developed as a promising strategy to treat HCC in the future.

Fig. 5  Apoptosis and necrosis were induced by MBRI-001 in HCC orthotopic mice model. The tumor tissues from control-treated group or MBRI-001-treated group were evaluated by H&E staining, IHC staining with the primary antibody against cleaved caspase-3, and TUNEL assays. Scale bar was 50 µm

Fig. 6  Combination studies in vitro and in vivo. a HCCLM3 cells were treated with MBRI001 for 72 h; sorafenib for 72 h; and co-treated by MBRI-001 and sorafenib (combination) for 72 h. The inhibition rates were measured by MTT assay. b Mice were treated with MBRI001 (6 mg/kg) intraperitoneally once 2 days, sorafenib (30 mg/ kg) orally once daily, combination of two agents, or vehicle alone once 2 days for 2 weeks. Tumor volume (mm3) was monitored once every third day. c Tumor weight was monitored at the end of experiment. P < 0.05 was considered statistically significance. **P < 0.01. d Body weight was monitored once every third day. Data were expressed as the mean ± SEM of five mice in eachgroup

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Cancer Chemotherapy and Pharmacology Funding  This study was funded by “Zhufeng Scholar Program” of Ocean University of China (841412016), “Major Projects of Independent Innovation” of Qingdao (15-4-13-zdzx-hy), “Outstanding Talents Plan” of Qingdao (15-10-3-15-(34)-zch), Aoshan Talents Cultivation Program of Qingdao National Laboratory for Marine Science and Technology (No.2017ASTCP-OS08) to Dr. Wenbao Li, National Natural Science Foundation of China (NSFC 81472687 and 81773761) to Dr. Linna Li, and youth special fund for PhD of Qingdao (No.16-5-1-62jch) to Dr. Jianchun Zhao.

Compliance with ethical standards  Conflict of interest  All authors declare that there is no conflict of interest. Ethical approval  All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

References 1. Akhmanova A, Steinmetz MO (2015) Control of microtubule organization and dynamics: two ends in the limelight. Nat Rev Mol Cell Biol 16(12):711–726. https​://doi.org/10.1038/nrm40​ 84 2. Wang Y, Yu Y, Li GB, Li SA, Wu C, Gigant B, Qin W, Chen H, Wu Y, Chen Q, Yang J (2017) Mechanism of microtubule stabilization by taccalonolide AJ. Nat Commun 8:1–8. https​:// doi.org/10.1038/ncomm​s1578​7 3. Chen J, Sun WL, Wasylyk B, Wang YP, Zheng H (2012) c-Jun N-terminal kinase mediates microtubule-depolymerizing agentinduced microtubule depolymerization and G2/M arrest in MCF-7 breast cancer cells. Anticancer Drugs 23(1):98–107. https​://doi.org/10.1097/CAD.0b013​e3283​4bc97​8 4. Wei RJ, Lin SS, Wu WR, Chen LR, Li CF, Chen HD, Chou CT, Chen YC, Liang SS, Chien ST, Shiue YL (2016) A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells. Toxicol Appl Pharmacol 311:88–98. https​://doi. org/10.1016/j.taap.2016.09.021 5. Kingston DG, Snyder JP (2014) The quest for a simple bioactive analog of paclitaxel as a potential anticancer agent. Acc Chem Res 47(8):2682–2691. https​://doi.org/10.1021/ar500​203h 6. Rutkauskiene G, Labanauskas L (2005) Treatment of patients of high-risk group of medulloblastoma with the adjuvant lomustine, cisplatin, and vincristine chemotherapy. Medicina (Kauas) 41(12):1026–1034 7. Nicholson B, Lloyd GK, Miller BR, Palladino MA, Kiso Y, Hayashi Y, Neuteboom ST (2006) NPI-2358 is a tubulindepolymerizing agent: in-vitro evidence for activity as a tumor vascular-disrupting agent. Anticancer Drugs 17(1):25–31 8. Honda-Uezono A, Kaida A, Michi Y, Harada K, Hayashi Y, Hayashi Y, Miura (2012) Unusual expression of red fluorescence at M phase induced by anti-microtubule agents in HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Biochem Biophys Res Commun 428(2):224– 229. https​://doi.org/10.1016/j.bbrc.2012.10.014 9. Bertelsen LB, Shen YY, Nielsen T, Stodkilde-Jorgensen H, Lloyd GK, Siemann DW, Horsman MR (2011) Vascular effects of plinabulin (NPI-2358) and the influence on tumour response when given alone or combined with radiation. Int J Radiat Biol 87(11):1126–1134. https​://doi.org/10.3109/09553​ 002.2011.60541​8

10. Singh AV, Bandi M, Raje N, Richardson P, Palladino MA, Chauhan D, Anderson KC (2011) A novel vascular disrupting agent plinabulin triggers JNK-mediated apoptosis and inhibits angiogenesis in multiple myeloma cells. Blood 117(21):5692– 5700. https​://doi.org/10.1182/blood​-2010-12-32385​7 11. Ding Z, Cheng H, Wang S, Hou Y, Zhao J, Guan H, Li W (2017) Development of MBRI-001, a deuterium-substituted plinabulin derivative as a potent anti-cancer agent. Bioorg Med Chem Lett 27:1416–1419. https​://doi.org/10.1016/j.bmcl.2017.01.096 12. El-Serag HB (2012) Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology 142(6):1264–1273. https​ ://doi.org/10.1053/j.gastr​o.2011.12.061 13. Xiao J, Lv Y, Jin F, Liu Y, Ma Y, Xiong Y, Liu L, Zhang S, Sun Y, Tipoe GL, Hong A, Xing F, Wang X (2017) LncRNA HANR promotes tumorigenesis and increase of chemoresistance in hepatocellular carcinoma. Cell Physiol Biochem 43(5):1926– 1938. https​://doi.org/10.1159/00048​4116 14. Alsaied OA, Sangwan V, Banerjee S, Krosch TC, Chugh R, Saluja A, Vickers SM, Jensen EH (2014) Sorafenib and triptolide as combination therapy for hepatocellular carcinoma. Surgery 156(2):270–279. https​: //doi.org/10.1016/j. surg.2014.04.055 15. Liang Y, Chen J, Yu Q, Ji T, Zhang B, Xu J, Dai Y, Xie Y, Lin H, Liang X, Cai X (2017) Phosphorylated ERK is a potential prognostic biomarker for Sorafenib response in hepatocellular carcinoma. Cancer Med. https​://doi.org/10.1002/cam4.1228 16. Villanueva A, LIovet JM (2011) Targeted therapies for hepatocellular carcinoma. Gastroenterology 140:1410–1426. https​:// doi.org/10.1053/j.gastr​o.2011.03.006 17. Wu FX, Chen J, Bai T, Zhu SL, Yang TB, Qi LN, Zou L, Li ZH, Ye JZ, Li LQ (2017) The safety and efficacy of transarterial chemoembolization combined with sorafenib and sorafenib mono-therapy in patients with BCLC stage B/C hepatocellular carcinoma. BMC Cancer 17(1):645–656. https​: //doi. org/10.1186/s1288​5-017-3545-5 18. Petrini I, Lencioni M, Ricasoli M, Iannopollo M, Orlandini C, Oliveri F, Bartolozzi C, Ricci S (2012) Phase II trial of sorafenib in combination with 5-fluorouracil infusion in advanced hepatocellular carcinoma. Cancer Chemother Pharmacol 69(3):773–780. https​://doi.org/10.1007/s0028​0-011-1753-2 19. Wild AT, Gandhi N, Chettiar ST, Aziz K, Gajula RP, Williams RD, Kumar R, Taparra K, Zeng J, Cades JA, Velarde E, Menon S, Geschwind JF, Cosgrove D, Pawlik TM, Maitra A, Wong J, Hales RK, Torbenson MS, Herman JM, Tran PT (2013) Concurrent versus sequential sorafenib therapy in combination with radiation for hepatocellular carcinoma. PLoS One 8(6):e65726. https​://doi.org/10.1371/journ​al.pone.00657​26 20. Xuan ZX, Li LN, Zhang Q, Xu CW, Dang DX, Yuan Y, An YH, Wang SS, Li XW, Yuan SJ (2014) Fully human VEGFR2 monoclonal antibody BC001 attenuates tumor angiogenesis and inhibits tumor growth. Int J Oncol 45(6):2411–2420. https​://doi. org/10.3892/ijo.2014.2690 21. Checchi PM, Nettles JH, Zhou J, Snyder JP, Joshi HC (2003) Microtubule-interacting drugs for cancer treatment. Trends Pharmacol Sci 24(7):361–365. https​://doi.org/10.1016/S0165​ -6147(03)00161​-5 22. Stefański T, Mikstacka R, Kurczab R, Dutkiewicz Z, Kucińska M, Murias M, Zielińska-Przyjemska M, Cichocki M, Teubert A, Kaczmarek M, Hogendorf A, Sobiak S (2018) Design, synthesis, and biological evaluation of novel combretastatin A-4 thio derivatives as microtubule targeting agents. Eur J Med Chem 144:797–816. https​://doi.org/10.1016/j.ejmec​h.2017.11.050 23. Fanale D, Bronte G, Passiglia F, Calò V, Castiglia M, Piazza FD, Barraco N, Cangemi A, Catarella MT, Insalaco L, Listì A, Maragliano R, Massihnia D, Perez A, Toia F, Cicero G, Bazan V (2015) Stabilizing versus destabilizing the

13



24. 25. 26. 27.

Cancer Chemotherapy and Pharmacology microtubules: a double-edge sword for an effective cancer treatment option?. Anal Cell Pathol 2015:1–19. https​: //doi. org/10.1155/2015/69091​6 Timmins GS (2014) Deuterated drugs: where are we now? Expert Opin Ther Pat 24(10):1067–1075. https ​ : //doi. org/10.1517/13543​776.2014.94318​4 Wassmann K, Benezra R (2001) Mitotic checkpoints: from yeast to cancer. Curr Opin Genet Dev 11(1):83–90. https​://doi. org/10.1016/S0959​-437X(00)00161​-1 Woods CM, Zhu J, McQueney PA, Bollag D, Lazarides E (1995) Taxol-induced mitotic block triggers rapid onset of a p53-independent apoptotic pathway. Mol Med 1(5):506–526 Li QQ, Hsu I, Sanford T, Railkar R, Balaji N, Sourbier C, Vocke C, Balaji KC, Agarwal PK (2017) Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and

13

xenografts via blockade of the cell cycle at G2/M. Cell Mol Life Sci. https​://doi.org/10.1007/s0001​8-017-2681-z 28. Yu CY, Jerry Teng CL, Hung PS, Cheng CC, Hsu SL, Hwang GY, Tzeng YM (2017) Ovatodiolide isolated from Anisomeles indica induces cell cycle G2/M arrest and apoptosis via a ROSdependent ATM/ATR signaling pathways. Eur J Pharmacol. https​://doi.org/10.1016/j.ejpha​r.2017.09.050 2 9. Cao H, Phan H, Yang LX (2012) Improved chemotherapy for hepatocellular carcinoma. Anticancer Res 32(4):1379–1386 30. Chen MC, Huang HH, Lai CY, Lin YJ, Liou JP, Lai MJ, Li YH, Teng CM, Yang CR (2015) Novel histone deacetylase inhibitor MPT0G009 induces cell apoptosis and synergistic anticancer activity with tumor necrosis factor-related apoptosis-inducing ligand against human hepatocellular carcinoma. Oncotarget 7(1):402–417. https​://doi.org/10.18632​/oncot​arget​.6352