Alteronol Induces Differentiation of Melanoma B16-F0 Cells

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116 Recent Patents on Anti-Cancer Drug Discovery, 2015, 10, 116-127

Alteronol Induces Differentiation of Melanoma B16-F0 Cells

Caixia Wang1, Bo Zhang

2, Na Chen

2, Liangliang Liu

2, Jinglei Liu

2, Qi Wang

2, Zhenhua Wang

3,

Xiling Sun1,*

and Qiusheng Zheng1,*

1Binzhou Medical University, Yantai 264003, P.R. China;

2Pharmacy School, Shihezi

University, Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of

Education, School of Pharmacy, Shihezi, 832002, P.R. China; 3Life sciences School, Yantai

University, Yantai, 264005, P.R. China

Received: March 25, 2014; Accepted: September 3, 2014; Revised: September 11, 2014

Abstract: Alteronol, isolated from microbial mutation strains, has been applied for Chinese and

International patents for tumor treatment. The aim of this project study is to investigate characteristics of proliferation and

redifferentiation induced by alteronol in B16-F0 mouse melanoma cells. Cell proliferation is determined by tetrazolium

salt colorimetric method (MTT assay). Morphological changes were analyzed by using Giemsa staining. The levels of

melanin and tyrosinase were measured by spectrophotometry. The mRNA expressions of tyrosinase-related protein Trp1

and Trp2 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The anchorage-independent pro-

liferation of B16-F0 was monitored by the colony formation assay. Tumorigenicity was characterized by an animal model

in vivo. The results showed that the proliferation of B16-F0 cells was inhibited by alteronol in a concentration and time

dependent manner. All well-known evaluation indexes of melanoma cell differentiation, including morphological changes

and tyrosinase activity alteration, were greatly enhanced with the increase of alteronol concentrations. Taken together, the

expression of tyrosinase related gene, decreased cell colony formation rate and the tumorigenicity in vivo; all of these re-

vealed that alteronol plays a key role in inducing differentiation and suppressing the proliferation of B16-F0 tumor cells

in vitro and in vivo.

Keywords: Alteronol, B16F0, differentiation, melanin, tyrosinase, tumorigenicity.

1. INTRODUCTION

Malignant melanoma is due to the dedifferentiation of

melanocytes that has been considered to be one of the most

common cancers presently. One characteristic feature of tu-

mors is the infinite proliferative potential [1], but at present

effects of chemotherapy treatments on anti-proliferation are

very limited, and tumor metastasis and recurrence occur

frequently [2, 3]. As effective methods for treatment of can-

cer are very few, the median survival time for people who

suffered from cancer is only six to ten months [4, 5]. There-

fore, it is urgent to find novel and low toxicity drugs as

effective treatment measures for malignant melanoma

through inducing tumor cells differentiation and it is also the

goal of every scientific research workers [6, 7].

Recently, several patents reported the ways of treatment

melanoma by inducing cells apoptosis or differentiation. The

patent US20130217949 [8] provides a method for inducing

melanoma tumor cells apoptosis by reducing Akt3 activity,

allowing a lower concentration of chemotherapeutic agents

to bring patients more decreased drug toxicity. Signaling

*Address correspondence to this author at the Binzhou Medical University,

Yantai 264003, P.R. China; Tel: +86 535-6913186; Fax: 0535-6913187;

E-mails: [email protected]; [email protected]

pathways normally connect extracellular signals to the nu-

cleus, regulating expression of genes that directly or indi-

rectly control cell growth, differentiation, survival and death.

Signaling pathways implicated in human oncogenesis in-

clude, but are not limited to, the Notch pathway, the Ras-

Raf-MEK-ERK or MAPK pathway, the PI3K-AKT pathway,

the CDKN2A/CDK4 pathway, the Bcl-2/TP53 pathway, and

the Wnt pathway. Notably, according to the patent

WO2013144923 MEK inhibitors and RAF inhibitors could

be used to treat melanoma [9]. Previous studies demonstrated

that antibodies to the human Notch ligand Delta-like ligand 4

(DLL4) can decrease the percentage of cancer stem cells or

tumor initiating cells in some xenograft tumors. These find-

ings suggest that targeting the Notch pathway, for example

with DLL4 antagonists, could help eliminate not only the

majority of non-tumorigenic cancer cells, but also the tu-

morigenic cancer stem cells responsible for formation and

recurrence of solid tumors. Lately, methods of treating mela-

noma and metastatic melanoma and of reducing the fre-

quency of tumor initiating cells (or cancer stem cells) in

melanoma tumors have been described in the patent

US20140065149 [10], which comprise administering a

DLL4 antagonist (e.g., an antibody that specifically binds the

extracellular domain of human DLL4) to a subject. Related

polypeptides and polynucleotides, compositions comprising

Qiusheng ZhengCaixia Wang

2212-3970/15 $100.00+.00 © 2015 Bentham Science Publishers

Alteronol Induces B16-F0 Differentiation Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 117

the DLL4 antagonists, and methods of making the DLL4

antagonists are also described elaborately.

Alteronol see Fig. (1), molecular weight of 351, is a new

type of compound which is isolated from a microbial muta-

tion strains (Alternaria alternata var. monosporus) that

derived from yew bark by the process of fermentation and

purification. Alteronol has been applied for the patent of

International (PCT/SG2005/000324) [11]. Our previous

studies have proved that alteronol can induce G1-phase ar-

rest and inhibit proliferation in Hela cells [12], and also can

induce apoptosis of B16-F0 cells through caspase 3 pathway

[13]. Furthermore, cell differentiation has a close relation-

ship with cell cycle arrest, which is usually accompanied by

abnormal cell cycle, for example HMBA can induce cell

cycle arrest associated with gastric epithelial cell differentia-

tion [14]. It is reported that G1 regulatory molecules have

been shown to be exquisitely regulated during the differen-

tiation process, exerting a key role in differentiation of many

cell models [16]. And the patent WO2008013918 [15] pro-

vides not only compositions and methods for regulating neu-

ral cell proliferation or differentiation, but also selecting ef-

fective bioactive agents for regulating proliferation or differ-

entiation.

So we hypothesize that alteronol could have an effect on

inducing cancer cells differentiation. To clarify the role of

alteronol on B16-F0 cells differentiation, the changes of

related factors involved in B16-F0 cells differentiation were

investigated. Our study aims to provide evidences for further

research and development of alteronol.

2. MATERIALS AND METHODS

2.1. Materials and Reagents

Alteronol with 99.5% purity was obtained from Shantou

Strand Biotech Co., Ltd. Dulbecco’s modified Eagle’s

medium (DMEM) and fetal bovine serum (FBS) were

obtained from Gibco Laboratories (Grand Island, NY, USA).

Penicillin and streptomycin were purchased from Shandong

Lukang Pharmaceutical Co., Ltd. (Shandong, China). L-

DOPA, Triton X-100, Thiazolyl blue (MTT) were obtained

from Sigma Chemical Co. (St. Louis, MO). All other

chemical reagents were of analytical grade and commercially

available.

2.2. Cell Culture

B16-F0 cells were obtained from China Center for Type

Culture Collection (Wuhan, China). The cells were cultured

at 37°C under a humidified 5% CO2 atmosphere in DMEM

supplemented with 10% FBS, and 100U/ml penicillin and

100μg/ml streptomycin. Logarithmically growing B16-F0

cells at a density of 1 105

cells/ml were used for each

experiment. Cells between passages 15 and 25 were used in

our study to avoid changes of cell characteristics during

extended cell culture periods.

H H

O OO

OH

H

O

HO

Fig. (1). Structure of alteronol.

2.3. Experimental Animals

Inbred male C57BL/6 mice at 6-8 weeks of age were

obtained from The Animal Center of Xinjiang Medical Uni-

versity (Urumqi, China, SCXK2003-0001). The mice were

housed under controlled temperature (22-26°C), humidity

(50-60%), and lighting (12h cycle), and standard feed and

water were provided ad libidum. Seven mice were used in

one experimental group, each group and experiments were

repeated at least twice.

2.4. Cell Proliferation Assay

Cells were incubated in 96-well plates in triplicate at

approximately 5 104 cells per well for 24h, and then treated

with alteronol (0, 0.4, 1.6, 2.4, 3.2, 4μg/ml) for 24h or 48h at

37°C. Cell viability was evaluated using MTT assay [17].

The absorbance was measured under a microplate reader

(Thermo Varioskan Flash 3001, USA) at 490nm.

2.5. Analysis of Morphological Changes

Treating B16-F0 cells for 48h with different concentra-

tions of alteronol from 0μg/ml to 3.2μg/ml after the cells

were seeded on coverslips adjusted to 1 105cell/ml. After

fixed by methanol, slides were stained for 20min with

Giemsa staining solution. When slides were rinsed in

deionized water and air-dried, then they were observed under

a phase-contrast microscopy (Zeiss) [18]. The stained cells

were assessed about their size and morphological character-

istics of the extended dendrites.

2.6. Determination of Melanin Content

Extracellular and intracellular melanin content were

measured according to the method described by Hill et al.

[19], B16-F0 cells were seeded and treated as described pre-

viously. After incubation at 37˚C in 5% CO2 for 48h, the

supernatant and cells was collected separately, and 1ml of

0.4M HEPES buffer (pH 6.8) and EtOH (9:1, v/v) was added

to 1ml of the medium. The OD (475nm) was measured to

quantify extracellular melanin; the calibration curve was

obtained with synthetic melanin solutions. B16-F0 cells

incubated in medium DMEM were collected and washed

118 Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 Wang et al.

twice with PBS and digested in 1ml of 1N NaOH for 16h at

room temperature for assessment of intracellular melanin.

2.7. Measurement of Tyrosinase Activity

For measurement of tyrosinase activity, we used the

method of L-DOPA oxidation as described previously [20].

After the cells were washed with ice-cold PBS, then cells

were lysed in lysis buffer containing 1% Triton X-100 and

PMSF (0.1mM) at -80°C for 30min, 80μl of supernatant and

20μl of L-DOPA were mixed for 40 min at 37°C in a 96-well

plate, then the absorbance at 475nm was measured in a

microplate reader (Thermo Varioskan Flash 3001, USA).

2.8. Evaluation of the mRNA Expressions of Tyrosinase-Related Protein Trp1 and Trp2

B16-F0 cells were treated with 0, 0.4, 0.8, 1.6, 2.4,

3.2μg/ml alteronol for 48h. Total RNA was isolated using

TRIZOL Reagent. The synthesis of cDNA was complished

in 25μl reaction solutions by using of 3μl total RNA primed

with oligod (T) (deoxy-thymidine). The cDNA templates

were amplified by PCR in a mix containing cDNA, 10 PCR

buffer, 2.5mM dNTPs, 10mM forward and reverse primers,

DNA polymerase, and sterile water. The sequence of the

tyrosinase primer was as follows: upstream 5'-GGC CAG

CTT TCA GGC AGA GGT-3', and downstream 5'-TGG

TGC TTC ATG GGC AAA ATC-3'; TRP-1 primer was as

follows: upstream 5'-GCTGCA GGA GCC TTC TTT CTC-3',

and downstream 5'-AAG ACG CTG CAC TGC TGG TCT-3';

TRP-2 primer was as follows: upstream 5’-GGA TGA CCG

TGA GCA ATG GCC-3', and downstream 5'-CGG TTG

TGA CCA ATG GGT GCC-3'. Primers specific for GAPDH

(upstream 5'-CAA GGT CAT CCA TGA CAA CTT TG-3'

and downstream 5'-GTC CAC CAC CCT GTT GCT GTA

G-3') were added as a control for the same reverse-

transcription product [21].

2.9. Soft Agar Colony Formation Assay

The anchorage-independent proliferation of B16-F0

melanoma cells was performed by the soft agar colony

formation assay [22, 23]. In brief, B16-F0 cells from log-

phase cultures were resuspended in DMEM containing

0.35% low melting agarose, and plated onto solidified 0.6%

agarose in six-well culture plates at a density of 1 104 cells

per dish. The cells were incubated at 37°C, 5% CO2 for two

weeks. The clones composed of more than 50 cells were

counted, and colony-forming efficiency was expressed as a

ratio of the number of clones to the total number of cells

plated.

2.10. Tumorigenicity Experiments

To further determine the tumorigenicity of differentiated

B16-F0 melanoma cells induced by alteronol, the tumori-

genicity experiment was employed. B16-F0 melanoma cells

were pretreated with alteronol for 48h and then washed

resuspended cells. Following this process, 6 105 cells in 200μl

DMEM were inoculated s.c. to syngeneic mice [24, 25].

Tumor sizes were measured every 2 days and were

calculated using the formula A B 0.52 (A, length; B,

width; all measured in millimetres). At the end of this

experiment, mice were sacrificed according to institutional

guidelines for the animals’ welfare [26].

2.11. Statistical Analysis

Data obtained from at least three independent

experiments were presented as means + S.E. and evaluated

by analysis of ANOVA followed by Student's t test. P < 0.05

was considered statistically significant.

2.12. Patient Consents & Animal Protection

The procedures were in accordance with the eighth edi-

tion of Guide for the Care and Use of Laboratory Animals.

All of the experiments were completed conform to the stan-

dard of the approved institutional experimental animal care

and use protocols.

3. RESULTS

3.1. Alteronol Inhibits the Proliferation of B16-F0 Cells

To determine the effect of alteronol on the inhibition of

proliferation in B16-F0 cells, the cells were treated with al-

teronol (0 to 4μg/ml). A significant inhibition in cell prolif-

eration was observed in a concentration and time-dependent

manner Fig. (2). After 48h and 24h treatment on B16-F0

cells, the calculated IC50 value was about 3.05μg/ml and

3.59μg/ml.

3.2. Alteronol Treatment Induces Morphological Changes in B16-F0 Cells

Morphological changes about dendrite outgrowth and

melanogenesis are considered as specific differentiation

markers of B16-F0 cells [27]. As shown in Fig. (3), after

treatment with alteronol, a clear morphological change was

confirmed; alteronol treatment induced the formation of

dendritic-like projections, which endowed treated cells with

star-like shape as compared to untreated cells. The altered

morphology was similar to that of neural cells because some

of the extended dendrites fused with other dendrites of

neighboring cells and had synapse-like knobs on their den-

drites. There is a further decrease in the number of cells,

which was more remarkable with the increase of treatment

concentrations.

3.3. Alteronol Treatment Induces Melanogenesis in B16-F0 Cells

After 48h incubation, melanogenesis induced by alteronol

in B16-F0 cells was shown in Fig. (4). Both the extracellular

see Fig. (4A) and the intracellular see Fig. (4B) melanin quan-

tity significantly increased in all tested groups as compared


Fig. (2). Effects of alteronol on B16-F0 cells proliferation. The inhibition rate was determined by MTT assay after 24h or 48h of incubation.

Data are presented as means + SEM from nine samples of three independent experiments. *P 0.05, **P 0.01 vs. vehicle control by

ANOVA followed by the Student-Newman-Keuls test.

Fig. (3). Cells were treated with 0, 0.4, 0.8, 1.6, 2.4 and 3.2μg/ml alteronol for 48h, and photomicrographs were taken using a digital video

camera. Micrographs were collected on a Zeiss microscope at 200 magnifications.


A

B

Fig. (4). Effects of alteronol on the extracellular (A) and intracellular (B) melanin synthesis in B16-F0 cells after treatment for 48h in vitro. Results are the mean + SD from three separate experiments. *P < 0.05, **P < 0.01 versus vehicle control-treated cells. with that of controls. These results also demonstrated that alteronol induced a differentiation program in B16-F0 cells since stimulation of melanogenesis is considered a well-known marker for differentiation of melanoma cells [28].

3.4. Alteronol Treatment Increases Tyrosinase Activity

Melanin biosynthesis is regulated by melanogenic enzymes such as tyrosinase-related proteins 1 and 2 [29]. Melanin is synthesized by the conversion of L-tyrosine into dopaquinone by tyrosinase, which is the rate-limiting step of melanin biosynthesis [30]. As shown in Fig. (5), after treat-

ment with alteronol, the activity of tyrosinase increased sig-nificantly compared with that of controls.

3.5. Alteronol Increases Expression of Melanin-Biosynthetic Genes

Melanin synthesis is regulated by a complex network of gene expressions and signalling pathways. The melanogenic enzymes tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) are thought to be the major enzymes in melanin biosynthesis [30]. In order to further clarify the mechanism of tyrosinase activation in-


duced by alteronol, the levels of melanogenesis-related proteins including tyrosinase, TRP-1, and TRP-2 were de-termined in B16-F0 cells after exposed to alteronol. As shown in Fig. (6), mRNA expressions of tyrosinase, TRP-1, and TRP-2 were enhanced in alteronol-treated cells. The results were consistent with the elevation of tyrosinase activ-ity and melanogenesis induced by alteronol.

3.6. Alteronol Inhibits Colony Formation of B16-F0 Cells

Colony formation assay is an in vitro cell survival assay based on the ability of a single cell to proliferate into a colony which is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo “unlimited” division [31]. The percent of colonies was calculated using the number of colonies formed in treated group divided by number of colonies formed in control. The colony-forming efficiency in soft agar of the B16-F0 melanoma cells was shown in Fig. (7A), and colo-nies observed in a microscope were shown in Fig. (7B). Al-teronol significantly decreased the size and the number of colonies in soft-agar. Alteronol inhibited colony formation in a dose-dependent manner, suggesting that alteronol was able to effectively decrease the tumorigenicities in vitro Fig. (7C).

3.7. Alteronol Pre-Treatment Reduces Tumorigenicity of B16-F0 Cells In vivo

Successful differentiation of B16-F0 cells induced by alteronol in vitro prompted us to examine the tumorigenicity of the pre-treated B16-F0 melanoma cells in vivo. The B16-F0 melanoma cells were treated by alteronol as described in the section of materials and methods, and then inoculated s.c.

at the flank of mice. Tumors became measurable at days 5-7 in the control group; however the alteronol pretreatment groups resulted in a delay in the time of tumor appearance. Growth curves indicated by tumor volume were shown in Fig. (8A). Picture of isolated tumors was shown in Fig. (8B), tumors resulted from alteronol-pretreated cells were signifi-cantly reduced in size Fig. (8B) and weight Fig. (8C). As depicted in Fig. (8D), it showed a typically clear difference in tumor-bearing mice which were treated with different concentrations of alteronol. The results of histological ex-aminations of melanoma tumors were demonstrated in Fig. (8E). Alteronol treatment induced prominent alterations con-cerning cell morphology and melanin content. Compared with the control group, cell morphology became more irregu-lar and the melanin content increased greatly. Results from Fig. (8) revealed that the tumorigenicity of the pretreated B16-F0 melanoma cells was markedly lower than that of control.

4. DISCUSSION

Essential characteristics of malignant tumor are continu-ous division and constant multiplication, so a crucial ap-praisal of induced differentiation is the inhibitory effect on the multiplication and tumorigenicities. Morphological changes about dendrite outgrowth and melanogenesis are considered to be specific evaluation indexes for differentia-tion of B16-F0 cells [27]. Stimulation of melanogenesis is considered as a well-known marker for differentiation of melanoma cells [28]. Tyrosinase activity and melanin con-tent are well-known molecular markers of melanoma cells differentiation [32, 33]. Our results showed that alteronol treatment could reduce the malignant characteristics of B16-F0 cells, but increased the properties of cell normalization.

Fig. (5). Effect of alteronol on cellular tyrosinase activity. Values were normalized based on the protein concentrations. The data are ex-pressed as percentages compared to the controls and are shown as the mean + SEM. *P < 0.05, **P < 0.01.


A B

Fig. (6). Effects of alteronol on the mRNA expression of tyrosinase, Trp1 and Trp2 in B16F10 melanoma cells. (A) The expression of ty-rosinase, Trp1 and Trp2 mRNA were analyzed by RT-PCR. (B) Relative expression is shown normalized to GAPDH in all cells, bars repre-sent means + SD of three independent experiments *p < 0.05, **p < 0.01, vs. control.

A


Fig. (7) contd….

B

C

Fig. (7). The anchorage-independent growth of B16-F0 cells were examined by soft agar colony formation assay. A. Colonies were photo-graphed; B. colonies were counted in a microscope, representative phase contrast images are shown; C. Bar graph showed the differences of colony formation among the six groups. Data were presented as mean + SD for three independent experiments. *P < 0.05, **p < 0.01 as com-pared to control.

��

��


A

B

C


Fig. (8) contd….

D

E

Fig. (8). The effect of alteronol pretreatment on the formation of tumor in C57BL/6 mice. Alteronol pre-treatment significantly reduces tu-

morigenicity of B16-F0 cells in vitro. B16-F0 melanoma cells were pretreated with 0, 0.8, 1.6, 3.2μg/ml alteronol for 48h prior to s.c. admini-

stration. Serial tumor volumes (A) were measured every two days tumor weights (B) were measured at the end of experiment. Values repre-

sent mean + SD, (*P < 0.05, **P < 0.01 vs. control group). C. Typical picture of isolated tumors. D. Representative images of tumor-bearing

mice E. Tumors from the tumor-bearing mice were sectioned at 4mm and were stained with H & E.


All these changes suggested that the B16-F0 cells were in-clined towards normalization, and confirmed that alteronol had the ability of inducing B16-F0 cells redifferentiation and impelling the cells reversion against the malignant pheno-type.

The current strategy for tumor treatment is to kill rapid proliferating tumor cells and resection, whose effects can slow down primary tumours, but usually transient, so tumor relapses frequently occur. Low degree of differentiation, slow proliferation rate and no sensibility to antimetabolites possessed by cancer stem cells have become main reasons for tumorigenesis, tumor infiltration and tumor recurrence [34, 35]. In 1980’s, differentiation-inducing therapy seems to be a promising approach which has become a popular topic in biomedical fields [36]. It is a new principle that cancer cells can be transformed into normal cells, which leads to growth retardation. Studies have shown that status of nearly 90% leukemia patients have been improved in the effect of differentiation treatment by retinoic acid and all-trans reti-noic acid, and the survival rate is more than 70% [37]. Fur-thermore, newly discovered histone deacetylase inhibitor SAHA can induce the differentiation of murine erythroleu-kemia cells and can inhibit the growth of breast cancer cells in a dose-dependent manner [38]. All these studies have pro-posed that induction of cell differentiation may be effective strategies for cancer treatment.

Malignant melanomas are tumors that are well known to respond poorly to treatment with chemotherapeutic reagents. Melanoma is the most aggressive form of skin cancer. At the present, there is no effective chemotherapy against invasive melanoma. Although some differentiation inducers like the phorbol ester and DMSO can also induce the differentiation of human melanoma cells, it is difficult to apply in the clini-cal because of their toxicity [39]. So it has become a new target that finding a novel and less toxic potentially candidate drugs for cancer therapy, which has no adverse drug reaction for adjuvant treatment of cancer alone or in combination.

In conclusion, induction of cellular differentiation serves as an important effective measure for tumor treatment. Since differentiation-dependent processes can mediate the expression of resistance to carcinogenesis and can induce cancer cells to revert to a benign state without losing their proliferation ability [40], cell-specific differentiation could become an alternative cancer therapy.

It has been previously reported that cell cycle arrest and cell differentiation are tightly linked processes. In our previ-ous studies, we found that alteronol was able to inhibit proliferation through inducing a G1-phase arrest in Hela cells [12]. However, cell cycle arrest has a close relationship with cell differentiation which is usually accompanied by irreversible cell cycle exit, for example, HMBA induces cell cycle arrest associated with gastric epithelial cell differentia-tion [15]. And in mammalian cells, signals that induce or

facilitate differentiation often do so through regulation of the cell cycle [41].

As a novel anticancer drug, alteronol can induce the dif-ferentiation of B16-F0 cells. These results suggest that alter-onol be a potent anticancer agent for malignant melanoma cells.

CURRENT & FUTURE DEVELOPMENTS

Our evidence clearly demonstrates the implication of alteronol-induced differentiation in B16-F0 melanoma cells. At present, several studies in terms of the effectiveness of alteronol have been reported: (i) to induce proliferation inhi-bition in human promyelocytic leukemia HL-60 cells [42], (ii) to induce cycle arrest in human promyelocytic leukemia (HL-60) cells [43], (iii) to induce proliferation inhibition in Hela cells [12]. Nevertheless, the results of this study show that alteronol can induce the differentiation of B16-F0 cells mainly through enhancing tyrosinase activity and inhibiting cell growth. A combination of these results provide us with more detailed information about alteronol and lay a founda-tion for further study of the effects of this natural products. Evidence based on this experiment shows that alteronol has a good effectiveness in the treatment of malignant melanoma. However, the feasibility of alteronol in the clinical applica-tion need to be further confirmed. Thus, a large number of experiments on drug safety evaluation need to be carried out to ensure the reliability of clinical research.

CONFLICT OF INTEREST

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Alteronol induces differentiation of melanoma B16-F0 cells”.

ACKNOWLEDGEMENTS

Funding for this research was supported by the Devel-opment of Major New Drugs of China (No. 2009ZX09103).

LIST OF ABBREVIATIONS

MTT = Methyl Thiazolyl Tetrazolium

RT-PCR = Reverse Transcription-Polymerase Chain Reaction

Trp-1 = Tyrosinase-Related Protein-1

Trp-2 = Tyrosinase-Related Protein-2

GAPDH = Glyceraldehyde-3-Phosphate Dehydrogenase

TYR = Tyrosinase


FBS = Fetal Bovine Serum

DMEM = Dulbecco’s Modified Eagle’s Medium

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Alteronol Induces Differentiation of Melanoma B16-F0 Cells

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