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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 1  |  Page : 182-187

Antitumor efficacy of lidamycin against human multiple myeloma RPMI 8226 cells and the xenograft in nonobese diabetic/severe combined immunodeficiency mice


1 Department of Histology and Embryology, Basic Medical College, Hebei United University, Hebei, Beijing, China
2 Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
3 The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Ministry of Health, Beijing, China

Date of Web Publication13-Apr-2016

Correspondence Address:
Yongzhan Zhen
#1 Tian Tan Xi Li, Beijing 100050
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.146093

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 > Abstract 


Aims: The aim of this study is to explore the antitumor efficacy of lidamycin (LDM) against human multiple myelomas (MM).
Materials and Methods: Human MM RPMI 8226 cells and the xenograft model in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were used to examine the antitumor activity of LDM.
Results: Notably, LDM markedly suppressed the growth of human MM RPMI 8226 xenograft in NOD/SCID mice. In vitro, there was a significant reduction in cell proliferation after treatment with LDM. The overall growth inhibition correlated with the increase of apoptotic cells. The apoptosis-related proteins including caspase-3, 7, and 9 were activated, and poly adenosine diphosphate-ribose polymerase was cleaved. Further investigation revealed that cellular Bcl-2 and survivin decreased, whereas the level of Bax increased in the LDM-treated cells.
Conclusions: LDM is highly effective against the growth of MM xenograft in NOD/SCID mice. The potent apoptosis.inducing effect of LDM may be mediated through caspase. and mitochondria.dependent pathway.

Keywords: Apoptosis, lidamycin, multiple myeloma, tumor inhibition


How to cite this article:
Zhen Y, Shang B, Liu X, Lin Y, Zhen Y. Antitumor efficacy of lidamycin against human multiple myeloma RPMI 8226 cells and the xenograft in nonobese diabetic/severe combined immunodeficiency mice. J Can Res Ther 2016;12:182-7

How to cite this URL:
Zhen Y, Shang B, Liu X, Lin Y, Zhen Y. Antitumor efficacy of lidamycin against human multiple myeloma RPMI 8226 cells and the xenograft in nonobese diabetic/severe combined immunodeficiency mice. J Can Res Ther [serial online] 2016 [cited 2019 Nov 17];12:182-7. Available from: http://www.cancerjournal.net/text.asp?2016/12/1/182/146093




 > Introduction Top


Multiple myeloma (MM) is a type of malignancy derived from plasma cells which are immune cells in bone marrow that produce antibodies. MM accounts for approximately 10% of all hematological malignancies.[1] Standard chemotherapy only leads to temporary remission in 40-60% patients. Median survival is <3 years. Although high-dose chemotherapy and stem cell transplantation have improved the rate of complete remission, however, some myeloma cells escape from the treatment and thus almost all MM patients experience relapse.[2] Therefore, new therapeutic agents are needed for this disease.

Lidamycin (LDM, also called C-1027), an enediyne antitumor antibiotic produced by Streptomyces globisporus C-1027,[3] contains an acid protein of 110 amino acid residues and a chromophore of novel enediyne structure, with the former serving as protecting protein while the latter as an active component to damage DNA.[4] The apoprotein and chromophore can be dissociated and reconstituted, and the biological activity of the rebuilt molecule is comparable to that of the natural one.[5] LDM shows extremely potent cytotoxicity toward cultured cancer cells and markedly inhibits the growth of transplantable tumors in mice and human cancer xenografts in nude mice.[6],[7],[8] LDM is also highly active to multidrug-resistant cancer cells.[9] In addition, LDM is a potent anti-angiogenesis agent with markedly anti-metastatic activity.[10] The potent efficacy of LDM was ascribed to its DNA strand-scission activity.[11] LDM is currently undergoing phase II clinical trials in China.

In our previous studies, LDM shows highly cytotoxicity to myeloma cells.[12],[13] To provide further support for this potency and its potential use in clinical therapy, the present study is set to investigate the antitumor efficacy of LDM both in vitro and in vivo with human MM RPMI 8226 cells and the MM xenograft in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Moreover, the mechanism of the highly potent apoptosis inducing effect of LDM was investigated.


 > Materials and Methods Top


Chemicals and reagents

Lidamycin was generously provided by Prof. Lian-Fang Jin (Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing, China). LDM stock solution (10 μM) was prepared in 0.9% NaCl and stored at −70°C. 5-fluorouracil (5-FU) was the product of Shanghai-Xudong Pharmacceutical Co. Ltd., (Shanghai, China). Dexamethasone (DEX) was obtained from Jiangsu-Suxin Pharmacceutical Co. Ltd., (Nanjing, JS, China). Adriamycin (ADM) was from Shenzhenwanle Pharmacceutical Co., Ltd., (Shenzhen, GZ, China). All other chemicals were of standard analytical grade.

Cell culture

Human MM cell lines (RPMI 8226, U266, and SKO-007) were cultured in RPMI-1640 (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Chemical Co., St. Louis, MO, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. RPMI 8226 cell line was obtained from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). SKO-007 and U266 and were kindly provided by Prof. Bei-Fen Shen (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China).

In vivo therapeutic effects

Twenty-Four female NOD/SCID mice (20 ± 2 g), obtained from the Institute of Experimental Animals, Chinese Academy of Medical Sciences (Beijing, China) at the age of 4-6 weeks, were used for human MM RPMI 8226 xenografts. Animal experiment was approved by the Animal Institutional Committee of our university and all animals received humane care. RPMI 8226 tumors for implantation were initially grown by injections of RPMI 8226 cells at a dose of 5 × 106 cells/mouse. A tumor piece of 2-3 mm in diameter was implanted subcutaneously into each experimental animal. After 9 days of tumor growth, the animals were randomly divided into groups (n = 6) in a manner that minimized the difference in tumor size between the groups. Each animal was given 200 μl of either phosphate buffer saline (PBS) (vehicle control) or LDM by intravenous injection. Second injection was given 12 days later. The doses of LDM were 0.02 mg/kg, 0.04 mg/kg, and 0.06 mg/kg, respectively. The mice were weighed, and tumor sizes were measured with a caliper and recorded every other day. Tumor volume was calculated using the formula: V = ab2/ 2. In this case, represents the length, and b is the width of the tumor mass in mm. Animals were killed when the tumors in the control group reached approximately 2 cm 3.

Western blotting analysis

Cells were harvested and washed with PBS. The whole cellular extracts were prepared by incubating cells on ice in lysis buffer containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 1% Nonidet P - 40, 0.1% sodium dodecyl sulfate (SDS), protease inhibitors (1 mM PMSF, 5 µg/ml aprotinin, 5 μg/ml leupeptin and 5 µg/ml pepstatin) and phosphatase inhibitors (20 mM [beta]-glycerophosphate, 50 mM NaF and 1 mM Na3 VO4. The cell lysates were cleared by centrifugation at 12,000 g for 12 min. Protein concentrations were determined by Bradford assay. Equal amounts of lysate (40 μg) were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA). Membranes were blocked in TBST containing 5% nonfat skim milk at room temperature for 2 h and probed with primary antibodies overnight at 4°C. Then membranes were blotted with an appropriate horseradish peroxidase-linked secondary antibody (Santa Cruz, CA, USA). Proteins were visualized using enhanced chemiluminescence western blotting detection reagents (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

Cell proliferation assay

Cell proliferation was performed as described previously using CellTiter 96® Aqueous NonRadioactive Cell Proliferation Assay (Promega, Madison, WI, USA). Briefly, cells at 10,000/well were plated in triplicate in 96-well flat bottom plates in 90 μl of culture medium without any anticancer drug. After 2 h incubation, triplicate wells were treated with anticancer drugs (10 μl) at various concentrations. Cells were exposed to 20 μl of the solution containing 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) and phenazine methosulfate (PMS) (MTS and PMS at the ratio of 20:1) for the last 4 h of 48 h cultures. The absorbance was read at 490 nm using a microplate reader (Thermo LabSystem, Franklin, MA, USA). The experiments were done at least thrice, each in triplicate.

Cell cycle analysis

Cells were collected and fixed in ice-cold 70% ethanol and stored at −4°C overnight. Samples were then washed twice in PBS and resuspended in a solution of propidium iodide (PI) (50 μg/ml) and RNase A (200 μg/ml) in PBS for 30 min at 37°C in the dark. The stained cells were filtered through a 40 μm gauze mesh, and the single-cell suspensions were analyzed on a fluorescence-activated cell sorter (Becton-Dickinson, Franklin Lakes, NJ, USA).

Fluorescein isothiocyanate-Annexin V/propidium iodide apoptosis assay

Cells were collected and resuspended in 200 μl binding buffer. Then 10 μl fluorescein isothiocyanate (FITC)-labeled enhanced Annexin V and 100 ng PI were added. Upon incubation in the dark (15 min, at room temperature or 30 min at 4°C), the samples were diluted with 300 μl Binding Buffer. Flow cytometry was carried out on an FACScan instrument (Becton-Dickinson, Franklin Lakes, NJ, USA) and data were processed by WinMDI/PC-software.

Staining with hoechst33342

RPMI 8226 cells were treated with control media or LDM at various concentrations for 48 h, and then the cells were collected. After being centrifuged and washed with PBS, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then washed with PBS. Hoechst 33342 (5 μg/ml) was added to the fixed cells, incubated for 10 min at room temperature. Then cells were spread on coverslips, and imaged using an Olympus IX-70 inverted fluorescent microscope (Center Valley, PA, USA).

Statistical analysis

Results are expressed as the means ± standard deviation (SD) efficacy was compared using the Student's t-test and differences between the means were considered to be significant when P < 0.05.


 > Results Top


Cytotoxicity of lidamycin to human multiple myeloma cells

The growth inhibitory effect of LDM in human MM cells (RPMI 8226, U266, and SKO-007) was examined with MTS Assay as described in materials and methods. Cells were cultured for 48 h [Figure 1] in the presence of various concentrations of LDM. Three human MM cell lines showed a decreased cell proliferation after treatment with the LDM. The IC50 value of LDM for RPMI 8226, U266, and SKO-007 cells were 0.5481 ± 0.0076 nM, 0.0575 ± 0.0015 nM and 0.1585 ± 0.0166 nM, respectively.
Figure 1: Growth inhibition of human multiple myeloma cells by lidamycin (LDM). Cells were exposed to LDM for 48 h and determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium). Data are from three independent experiments

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Cytotoxicity of lidamycin and other drugs to RPMI 8226 cells

The growth inhibitory effect of LDM in RPMI 8226 cells was examined with MTS Assay as described in materials and methods. Cells were cultured for 48 h [Figure 2]a in the presence of various concentrations of LDM. Meanwhile, some conventional therapeutic agents including ADM, 5-FU, and DEX were also examined by the assay. RPMI 8226 cells showed decreased cell proliferation after treatment with the drugs, especially the cells exposed to LDM. The IC50 value of LDM for RPMI 8226 cells was 0.5481 ± 0.0076 nM, whereas those of ADM, 5-FU, and DEX were 25.1 ± 1.7 nM, 562.3 ± 25.3 nM, and 17783 ± 401 nM, respectively. In terms of IC50 values, the cytotoxicity of LDM was much more potent than that of other tested drugs. Determined by MTS Assay, RPMI 8226 cells treated with indicated concentrations of LDM (0.01, 0.1, 0.5, and 1 nM) for the indicated time showed a decreased cell proliferation in a time-dependent manner [Figure 2]b.
Figure 2: Growth inhibition of RPMI 8226 cells by lidamycin (LDM) and other drugs. (a) Cells were exposed to the drugs for 48 h and determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS). (b) Cells were cultured with LDM (0.01, 0.1, 0.5, and 1 nM) for the indicated time and determined by MTS. Data are from three independent experiments. 5-FU = 5-fluorouracil, DEX = Dexamethasone, ADM = Adriamycin

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Inhibition of human multiple myeloma xenograft growth in nonobese diabetic/severe combined immunodeficient mice

Treatment was started on day 9 after tumor transplantation. LDM was given at doses of 0.02 mg/kg, 0.04 mg/kg, and 0.06 mg/kg respectively through the caudal vein. On day 21, LDM was given for the 2nd time. Control mice were given PBS vehicle only. The growth of tumors in LDM-treated NOD/SCID mice was suppressed dose-dependently compared with the controls [Figure 3]. Treatment with LDM at the dose of 0.02, 0.04 mg/kg, and 0.06 mg/kg inhibited the growth of human MM RPMI 8226 xenografts by 68.1%, 82.4%, and 90.8%, respectively [Figure 3] and [Table 1]. The body weights of the animals showed no significant differences between the control and treated groups. These findings suggest that LDM at well-tolerated doses markedly inhibited tumor growth.
Figure 3: Effects of lidamycin (LDM) on the growth of human multiple myeloma RPMI 8226 xenograft in nonobese diabetic/severe combined immunodeficient mice. A total of two injections of LDM was administered by intravenous route on day 9 and day 21 after tumor transplantation

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Table 1: Inhibitory effects of LDM on the growth of human multiple myeloma RPMI 8226 xenografts in NOD/SCID mice

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Induction of apoptosis by lidamycin in RPMI 8226 cells

By Hoechst 33342, staining, the nuclei of untreated cells were normal in appearance and showed diffused staining of the chromatin. After exposure to LDM for 48 h, most cells presented typical morphological changes of apoptosis such as chromatin condensation or a shrunken nucleus [Figure 4]a. Apoptosis induced by LDM was further confirmed by FITC-Annexin V/PI staining. LDM at 0.1 nM induced earlier apoptosis in RPMI 8226 cells. The ratio of apoptosis was significantly enhanced when cells were incubated with 1 nM LDM for 48 h. It suggested that apoptosis was the predominant mode of LDM-induced cell death [Figure 4]b.
Figure 4: Induction of apoptosis by lidamycin (LDM) in RPMI 8226 cells. Cells were treated with LDM for 48 h at the indicated concentrations. (a) Cells stained with DNA-binding dye, Hoechest 33342. Photographs were taken under ×400 fluorescent microscope. (b) Cells labeled with a combination of fluorescein isothiocyanate-Annexin V and propidium iodide and followed by flow cytometric analysis. The percentage of apoptotic cells (lower right quadrant) is indicated

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Induction of cell cycle arrest by lidamycin in RPMI 8226 cell

Cells cultured for 48 h with control media or various concentrations of LDM (0.1 nM, 0.5 nM, and 1 nM). PI staining showed that LDM at 0.1 nM and 0.5 nM induced G2/M arrest in RPMI 8226 cells. There appeared an S phase arrest after treatment with 1 nM LDM [Figure 5].
Figure 5: Cell cycle analysis of cells treated with lidamycin (LDM). Cells were stained with propidium iodide after a 48 h and exposure to different concentrations of LDM. Percentages of cells in different phases of the cell cycle were determined by flow cytometric analysis

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Downregulation of Bcl-2 and Survivin in lidamycin-induced caspase-dependent apoptosis in RPMI 8226 cell

In order to investigate the role of apoptosis, the activity of caspases in response to LDM treatment was determined. Activation of various caspases can be detected by the appearance of a proteolytically cleaved, smaller molecular weight form of the proteins. As shown in [Figure 6], the cleaved caspase-3, caspase-7 and caspase-9 were activated by LDM in a dose dependent manner. In addition, poly adenosine diphosphate-ribose polymerase (PARP), one of the main substrates of activated caspase pathways and a well-established indicator of apoptotic cell death, was also cleaved in response to LDM treatment in a dose dependent manner. As caspase/PARP cleavage is correlated with LDM induced cytotoxicity, we next examined whether LDM treatment modulates the levels of anti-apoptosis proteins. As shown in [Figure 6], expression of Bcl-2 and survivin was markedly downregulated by LDM treatment in RPMI 8226 cells, whereas, the level of Bax was increased. These results suggest that the decrease of Bcl-2/Bax ratio and survivin expression plays a role, at least in part, in human MM cell apoptosis triggered by LDM.
Figure 6: Cells were cultured with lidamycin at different concentrations for 48 h and then lysed and subjected to western blotting as described in materials and methods. Casp = Caspase; CF = Cleaved fragment

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 > Discussion Top


Over the last several years, significant insight into the dysregulation of various signal transduction pathways of MM has led to the development of new agents which are expected to offer a better disease control. Two new drug classes have markedly changed the management of MM. Thalidomide analogs [14],[15],[16] and bortezomib [17],[18],[19] are promising in the treatment of MM. However, for treatment of this intractable disease to explore new effective therapeutic agents is urgently needed. LDM is a member of the enediyne antibiotic family.[12] It is undergoing phase II clinical trials in China. In our previous studies, LDM shows highly potent cytotoxicity to myeloma cells including U266, SKO-007 and SP2/0 cells.[8],[12] However, the in vivo therapeutic efficacy of LDM on MM xenograft has not been evaluated. As known, the human MM xenografts are useful models for testing the therapeutic effects of antitumor agents in vivo.[20],[21],[22] In the present study, LDM exerted significantly dose-dependent inhibition of the growth of MM RPMI 8226 xenograft in NOD/SCID mice. There was no significant body weight loss in the treated groups compared with the control group. All animals survived the duration of the experiment. The results indicate that LDM shows high efficacy against MM.

In terms of IC50 values, LDM showed much more potent cytotoxicity than conventional anti-MM agents to RPMI 8226 cells. LDM displays highly potent apoptosis-inducing effect in RPMI 8226 cells. It is evident that the induction of apoptosis appears to be the predominant mode of LDM-induced cell death. Moreover, LDM caused a progressive accumulation of MM cells in G2/M and S phase arrest. Mechanistically, LDM appears to trigger MM cells apoptosis by increasing the levels of cleaved caspase-3, caspase-7, caspase-9, and PARP. These caspases belong to the family of cystein proteases whose activation induces cellular apoptosis. Specifically, proteolytical cleaved caspase-3 and caspase-7, the active form of pro-caspase-3 and pro-caspase-7, are key molecules for identifying the activation of apoptosis.[23] Moreover, caspase-9, the upstream of caspase-3, caspase-7, is also activated which suggests the apoptosis induced by LDM is a mitochondrion-mediated pathway. In addition, PARP, one of the main substrates of activated caspase pathways and a well-established indicator of apoptotic cell death, is also cleaved by LDM in a dose-dependent way. Furthermore, the expression of anti-apoptosis proteins before and after LDM treatment was determined. As known, members of Bcl-2 protein family act as key regulators of cellular apoptosis and are important determinants of cellular sensitivity or resistant to chemotherapy drugs.[24],[25] Over-expression of Bcl-2, an anti-apoptosis member of this family, is commonly observed in human MM, and Bcl-2 over-expression correlates with chemo-resistance in this disease. In addition, survivin is a member of the inhibitor family of apoptosis that regulate caspase activity. Survivin plays a crucial role in the regulation of apoptosis. Down-regulation of survivin significantly inhibits tumor cell growth.[26],[27] The expression of Bcl-2 and survivin was significantly down-regulated by LDM in a dose-dependent way; whereas, the level of Bax was increased. As reported, a decrease of Bcl-2/Bax is sufficient to promote apoptosis in mammalian cells and induces cell death by activating the mitochondrial apoptosis in the upstream of caspase-9 and the latter naturally activates caspase-3.[28] In the present study, Bcl-2/Bax ratio and the level of survivin were decreased, and caspase-3, 7, 9, and PARP were activated with LDM treatment. Thus, the decrease of Bcl-2/Bax ratio and survivin expression might play a role, at least in part, mediating RPMI 8226 cell apoptosis triggered by LDM.

Taken together, the present study confirms that LDM, an enediyne antitumor antibiotic with extremely potent cytotoxicity to MM cells in vitro, exerts high efficacy against human MM xenograft in vivo. The study results provide a rationale for clinical trials of LDM to improve therapeutic outcome in MM.

 
 > References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
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