Research ArticleExperimental Studies

Suppressive Effects of Anisomycin on the Proliferation of B16 Mouse Melanoma Cells In Vitro

HIRONORI USHIJIMA, RINA MONZAKI and ARISA ONODERA
Anticancer Research December 2021, 41 (12) 6113-6121; DOI: https://doi.org/10.21873/anticanres.15431

Abstract

Background: Anisomycin, a potential anticancer therapeutic drug, exerts an antitumor effect on melanoma cells at a lower concentration than that required for other cancer cells. However, the molecular mechanisms remain unclear. Materials and Methods: The sensitivity to and cytotoxicity of anisomycin, as well as the effects of anisomycin on glucose metabolism and relative mRNA expression of senescence- and cancer-associated genes, were studied using B16 mouse melanoma cells. Results: The viability of anisomycin-treated cells decreased in a concentration-dependent manner, and the growth of cell spheroids was suppressed by 50 nM anisomycin. Glucose metabolism was reduced by anisomycin treatment, and the mRNA expression of genes responsible for growth inhibition, such as p21, p53 and Txnip was upregulated. Conclusion: The results suggest that anisomycin may be a promising future anticancer drug that is effective at low concentrations against melanoma by reducing glucose metabolism, causing cell senescence-like phenomena.

Key Words:

Melanoma, which is caused by the cancerous growth of melanocytes, is a highly lethal form of skin cancer, and exposure to ultraviolet light is thought to be one of the main causes of melanoma (1-3). Surgical procedures alone cannot cure metastatic melanoma and are, therefore, used in combination with drug therapy. Currently, molecular-targeted therapy using vemurafenib, which inhibits mutated v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), is often prescribed for the treatment of melanoma (4, 5). Nivolumab, an immune checkpoint inhibitor antibody against programmed cell death protein 1 (PD-1), is also used for the treatment of melanoma, which commonly overexpresses PD-1 ligand (PD-L1/2) to effectively turn off the immune response and evade immune surveillance (6). However, some patients do not respond to these drugs, other patients develop secondary resistance, and therefore, other drug targets for melanoma have been investigated.

Anisomycin (7) has been shown, in many in vivo and in vitro studies to have a range of biological activities, such as the inhibition of protein synthesis (8); activation of mitogen activated protein kinases (MAPKs), including c-Jun N-terminal kinase (JNK) (9), and the induction of ribotoxic (10) and oxidative stress (11). Anisomycin was recently reported to show in silico affinity for the main severe acute respiratory syndrome coronavirus 2 protease and could be a drug candidate to manage coronavirus disease 2019 (COVID19) (12). Anisomycin is also considered a potential anticancer therapeutic drug, and the molecular mechanisms of its antitumor effects have been widely studied in ovarian cancer, leukaemia, non-small-cell lung cancer and colorectal cancer (13-16).

A study on melanoma has reported that the combination therapy with anisomycin and lexatumumab, an agonistic human monoclonal antibody against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2, was more effective against human melanoma cells than single-agent treatment, owing to the activation of the apoptosis pathway (17). It has been reported that mouse melanoma cells expressing activating transcription factor (ATF)-derived peptide were sensitized to TRAIL mediated apoptosis by anisomycin (18). It has also been reported that the activation of the p38a (also known as MAPK14) pathway by anisomycin showed tumor-suppressive effects on patient-derived melanoma cells with mutant neuroblastoma RAS viral oncogene homologue (NRAS) (19). However, the molecular mechanisms by which anisomycin exerts an antitumor effect on melanoma cells at lower concentrations than that required for other cancer cells remain unclear. In the present study, we aimed to elucidate the mechanism of the suppressive effect of anisomycin on the proliferation of melanoma cells.

Materials and Methods

Reagents and cell culture. Minimum essential medium (MEM), trypsin, and foetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dimethyl sulfoxide and anisomycin were supplied by Nacalai Tesque, Inc. (Kyoto, Japan) and FUJIFILM Wako (Osaka, Japan), respectively. Trypan blue was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Three-dimensional (3D) Petri dish micromolds (20) were purchased from MicroTissues, Inc. (Providence, RI, USA). All other chemicals were of the highest commercially available grade.

B16 mouse melanoma cells were obtained from JCRB Cell Bank (Osaka, Japan) and checked to be free of mycoplasma contamination. Cells were cultured in MEM containing 10% (v/v) FBS and antibiotics (0.07 g/l penicillin G and 0.1 g/l streptomycin) at 37°C in a humidified atmosphere with 5% CO2. The proliferation of B16 cells in the presence of anisomycin was monitored by cell counting using a TC10 automated cell counter (Bio-Rad).

Measurement of cell viability and cytotoxicity of anisomycin. Cell Counting Kit-8 (CCK-8) with water-soluble tetrazolium salts (WST-8; Dojindo, Kumamoto, Japan) was used to measure the viability of anisomycin-treated B16 cells as per the manufacturer’s protocol. The stock solution of anisomycin was prepared in dimethyl sulfoxide. Briefly, cells were seeded into a 96-well microplate (6×103 cells/well). After 24 h, the medium was changed to a fresh medium containing different concentrations of anisomycin, followed by further incubation for 24 h. The CCK-8 solution (10 μl) was added to each well of the 96-well microplate, followed by incubation for 3 h at 37°C in a 5% CO2 incubator. Absorbance was measured at 490 nm using an iMark microplate reader (Bio-Rad). The cytotoxicity of anisomycin towards B16 cells was measured using a trypan blue dye exclusion assay (21). B16 cells treated with different concentrations of anisomycin for 24 h were harvested using 0.25% trypsin. An aliquot of the cell suspension stained with 0.4% trypan blue (Bio-Rad) was transferred to a cell counting slide, and the ratio of the living and dead cells was measured using a TC10 automated cell counter.

3D spheroid culture of B16 cells. Agarose gel microwells (1% agarose and 0.9% NaCl) were created using 3D Petri dishes as per the manufacturer’s protocol and then transferred to a 24-well plate and incubated with 1 ml of MEM for 1 h. B16 cells (4×103 cells) were seeded onto the microwells and incubated with 1 ml of MEM for 72 h at 37°C in a 5% CO2 incubator. Subsequently, the medium was changed to a fresh medium with or without 50 nM anisomycin. The cells were grown in the form of spheroids for 15 days, with the medium changed every 5 days. The shape of the spheroids was observed every 2 days using an Olympus IX71 microscope (Olympus, Tokyo, Japan). The perimeter of the spheroids was measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Measurement of glucose and lactate concentrations. B16 cells (6×103 cells/well) were seeded into a 96-well microplate and precultured for 24 h at 37°C in a 5% CO2 incubator. Subsequently, the medium was changed to a fresh medium with or without 50 nM anisomycin, and the cells were cultured for an additional 24 h. The cell supernatant was transferred to a 96-well plate, and the concentrations of glucose and lactate were determined using the glucose assay kit-WST (Dojindo) and lactate assay kit-WST (Dojindo), respectively. Absorbance was measured at 450 nm using an iMark microplate reader (Bio-Rad), and the concentrations of glucose and lactate were calculated based on calibration curves.

Measurement of intracellular NAD/NADH concentrations. NAD/NADH concentrations were determined using a NAD/NADH assay kit (Dojindo). In brief, B16 cells (1.2×105 cells) were seeded on a 100-mm dish and precultured for 24 h at 37°C in a 5% CO2 incubator. Subsequently, the medium was changed to a fresh medium with or without 50 nM anisomycin. After 24 h, the cells were harvested with 0.25% trypsin treatment and cell lysates were prepared using a NAD/NADH extraction buffer. The lysates were transferred to 10-kDa molecular weight cut-off filtration tubes and centrifuged at 12,000×g for 10 min. The filtrates were collected and divided into two samples each to determine NAD/NADH and NADH, respectively. After the sample solutions were reacted with a WST–enzyme working solution, the amounts of total NAD/NADH and NADH in the sample were calculated by measuring the absorbance at 450 nm using an iMark microplate reader (Bio-Rad).

Quantification of mRNA expression. B16 cells were cultured in MEM with or without 50 nM anisomycin. After 24 h, total RNA was extracted from control and anisomycin-treated cells using the RNeasy mini kit (Qiagen, Hilden, Germany). The total RNA (1 μg) was converted to cDNA using reverse transcription with the ReverTra Ace qPCR RT master mix (TOYOBO, Osaka, Japan). mRNA expression was quantified using the real-time quantitative polymerase chain reaction (RT-qPCR) method with an Eco real-time PCR system (Illumina, San Diego, CA, USA). The relative expression of each mRNA was calculated using the 2−∆∆Ct method (22), and the expression level was normalized to that of mouse β-actin (Actb) gene. The sequences of the primer sets used in this assay are listed in Table I.

View this table:
Table I.

Primer sets used in RT-qPCR.

Statistical analyses. Statistical analyses were conducted using R programming scripts. One-way analysis of variance with Bonferroni correction was used for the analysis of the cell viability, cytotoxicity and 3D spheroid culture assay data. The Student’s t-test was performed to determine significance of differences between two groups (control group vs. anisomycin-treated group; n=3 per group) in the glucose, lactate, and NAD/NADH assays. Differences in the relative expression of mRNA between the control and anisomycin-treated groups were also tested for statistical significance using the Student’s t-test. p<0.05 was considered to be statistically significant.

Results

Suppressive effects of low-dose anisomycin on the proliferation of B16 melanoma cells. A cell viability assay was first performed to determine the effective concentration of anisomycin against B16 mouse melanoma cells, and the cell viability was found to be comparable to that of the control cells, below 10 nM (Figure 1A). At concentrations of anisomycin higher than 10 nM, the cell viability decreased in a concentration-dependent manner and was below 50% at concentrations higher than 40 nM. The cytotoxicity of anisomycin to the control cells was found to be approximately 6%, and there were no statistically significant differences between the control cells and anisomycin-treated cells at concentrations of 80 nM or lower (Figure 1B). The highest cytotoxicity (approximately 16%) towards B16 cells was shown by anisomycin at a concentration of 100 nM.

Figure 1.
Figure 1.

Effects of anisomycin on the proliferation of B16 cells. (A) Cell viability after 24 h of treatment with the indicated concentrations of anisomycin. (B) Cytotoxicity of anisomycin at the indicated concentrations towards B16 cells after 24 h of treatment. Values represent mean±standard deviation (n=3). ***p<0.005; one-way analysis of variance with Bonferroni correction, compared to 0 nM anisomycin group.

Suppressive effects of anisomycin on the growth of B16 cell spheroids. Next, the suppressive effect of anisomycin on B16 cells was examined under 3D culture conditions. The size of the spheroid formed by control cells increased over time (Figure 2A) and on day 9, was roughly double compared to day 1. However, the size of the spheroid formed by cells treated with 50 nM anisomycin remained almost the same from day 1 to 15, indicating that the growth was suppressed by anisomycin. Measurement of the spheroid perimeters using the ImageJ software established that there were significant differences in the sizes of spheroids between control and anisomycin-treated cells after day 11 (Figure 2B, p=0.008 on day11, 0.00068 on day 13 and 0.00028 on day 15).

Figure 2.
Figure 2.

Growth of spheroids from control and anisomycin-treated B16 cells. (A) Photographs of spheroids at the indicated time points for control cells (upper panel) and anisomycin (50 nM)-treated cells (lower panel). (B) Spheroid perimeters were measured from the photographs in (A) using the ImageJ software. Values are the mean±standard deviation (n=3). **p<0.01, ***p<0.005; one-way analysis of variance with Bonferroni correction, compared to control group.

Inhibition of glucose metabolism by anisomycin treatment. To determine whether anisomycin alters glucose metabolism in B16 cells, glucose concentrations were compared in the culture media derived from B16 cells cultured in the presence or absence of 50 nM anisomycin. The data showed that the glucose concentration in the medium of anisomycin-treated cells was 1.7 times higher than that in the medium of control cells (Figure 3A). The concentration of lactate, which is produced as a result of glucose metabolism, was also quantified in the culture media. The results showed that the amount of lactate produced by the anisomycin-treated cells was reduced to one-third of that produced by the control cells (Figure 3B). In addition, intracellular NAD, which is one of the leading indicators of mitochondrial activity, was quantified in the control and anisomycin-treated groups of B16 cells. The results showed that anisomycin decreased the amount of intracellular NAD by 58%, whereas no significant difference was found in the amount of intracellular NADH (Figure 3C and D).

Figure 3.
Figure 3.

Effects of anisomycin on glucose metabolism in B16 cells. (A, B) Concentrations of glucose and lactate in the media after culturing cells with and without 50 nM anisomycin for 24 h. (C, D) Concentrations of total NAD/NADH and NADH in the lysates of control and anisomycin-treated cells. Values are the mean±standard deviation (n=3). ***p<0.005; Student’s t-test, compared to control group.

Anisomycin-caused changes in mRNA expression of senescence- and cancer-associated genes. Based on the intracellular NAD measurement, as shown in Figure 3, it was suggested that anisomycin might inhibit mitochondrial activity, and the decrease in the amount of NAD might induce senescence in B16 cells. Changes in the expression of senescence-associated genes, such as p16, p21, p53, and Sirt1 (sirtuin 1), were examined using RT-qPCR. No expression of p16 was detected, as the respective exon region might be deleted in the genome in B16 cells (23, 24). However, the relative mRNA expression levels of the other three genes were compared between control and anisomycin-treated cells. The relative mRNA expression levels of p21, p53, and Sirt1 were all higher in the anisomycin-treated cells compared to control cells. The fold changes were as follows: p21, 2.0; p53, 2.5 and Sirt1, 2.7 (Figure 4A). We next examined whether anisomycin altered the expression levels of tumor promoting genes and tumor suppressor genes in B16 cells. The relative mRNA expression levels of tumor promoting genes such as Egr1, Fos, Nfkb, and Tfrc in anisomycin-treated cells were compared with those in the control (Figure 4B). The relative expression levels of Egr1 and Nfkb were found to be 2.2- and 2.0-fold higher, respectively, in anisomycin-treated cells. However, the relative expression level of Fos was almost the same as that in the control, and the relative expression level of Tfrc was reduced to 60% of that in the control. Regarding the relative expression levels of tumor suppressor genes, Atf3, Klf6, and Txnip, the following fold changes were as following: Atf3, 0.99; Klf6, 0.93 and Txnip, 6.5 (Figure 4C).

Figure 4.
Figure 4.

Expression of senescence- and cancer-associated genes in untreated and anisomycin-treated B16 cells. Relative expression levels of senescence-related genes (A), oncogenes (B), and tumor suppressor genes (C). Values are the mean±standard error (n=3). *p<0.05, **p<0.01, ***p<0.005 (Student’s t-test, compared to control group).

Discussion

In this study, we examined the antitumor effects of anisomycin on B16 mouse melanoma cells, focusing on cell proliferation, glucose metabolism and changes in the expression of cellular senescence- and cancer-associated genes.

It was found that anisomycin at concentrations above 40 nM reduced the viability of B16 cells below 50%. The results of the trypan blue assay suggested that this growth inhibitory effect of anisomycin was not due to its cytotoxicity. Similar to the two-dimensional (2D) culture results, anisomycin suppressed spheroid enlargement of B16 cells in a 3D experimental system. Based on previous reports, 3D culture of cancer cells is considered to better mimic their behaviour in vivo than 2D culture (25). Our results suggest that anisomycin may have an antitumor effect on metastatic melanoma in vivo. The changes in intracellular metabolism and gene expression induced by anisomycin in 3D culture should be the subject of further investigation.

The tumor cell growth is thought to be closely related to intracellular energy metabolism, especially glucose metabolism (26). The best-known phenomenon is the Warburg effect, which refers to preferential use of aerobic glycolysis by many cancer cells, unlike normal cells, to obtain energy from glucose (27). We have previously reported that anisomycin altered intracellular metabolic pathways in human colorectal cancer cells (28). In this study, the residual glucose concentration in the medium of B16 cells treated with anisomycin was higher than that in the control, which suggested that glucose metabolism in B16 cells was reduced by anisomycin. It has been reported that inhibition of glucose uptake effectively reduces the metastatic potential of melanoma (29), which suggests that anisomycin may also have an inhibitory effect on melanoma metastasis.

Recently, the role of NAD metabolism in various types of cancer cells has attracted attention, and it has been reported that depletion of intracellular NAD leads to a decrease in the amount of ATP, resulting in a loss of plasma membrane homeostasis, followed by oncosis-mediated cell death (30). In melanoma cells, knockdown of indoleamine 2,3-dioxygenase 2, a tryptophan-catabolising enzyme, has been reported to reduce intracellular NAD and suppress cell proliferation, whereas the addition of exogenous NAD weakened growth inhibition (31). In the present study, we considered that the decrease in intracellular NAD levels was one of the reasons for the inhibitory effect of anisomycin on the proliferation of B16 cells.

The decrease in intracellular NAD has been reported to contribute to cell senescence (32), and therefore, effects of anisomycin on the expression levels of cell senescence- and cancer-associated genes were investigated. Increased expression of p21 and p53 is thought to be one of the characteristics of cellular senescence and is known to contribute to the inhibition of cell proliferation via cell cycle arrest (33). It was found that the mRNA expression levels of p21 and p53 were 2.0- and 2.5-fold higher, respectively, in B16 cells treated with anisomycin than in controls. The expression of SIRT1, also known as a longevity gene, generally decreases with cellular senescence (34) but was increased in B16 cells treated with anisomycin. Because SIRT1 is also known to be expressed in response to oxidative stress (35), the upregulation of Sirt1 by anisomycin treatment might have been a protective response. Increased mRNA expression of Egr1 and Nfkb, known tumor promoting genes, was observed in B16 cells treated with anisomycin. We considered that these increases might have been compensatory responses to the inhibitory effect of anisomycin on cell proliferation. The decrease in the mRNA expression of Tfrc could contribute to the suppression of B16 cell proliferation. Among the tumor suppressor genes (Atf3, Klf6, and Txnip) tested, the expression level of Txnip in B16 cells treated with anisomycin was 6.5-fold higher than that in the control. TXNIP is considered to be a potential tumor suppressor gene, and its expression levels are reduced in various cancer tissues (36, 37). Moreover, in vitro proliferation, metastasis, and invasion of cancer cells in which TXNIP is overexpressed are suppressed, and cell apoptosis is promoted (38). In melanoma cells, TXNIP is also considered to contribute to the suppression of cancer growth and metastasis (39). A potential mechanism for the anisomycin-mediated suppression of the proliferation of B16 cells may be inhibition of glucose uptake due to increased expression levels of Txnip (40).

It has been previously reported that low-dose anisomycin enhances lexatumumab-induced apoptosis and inhibits the synthesis of antiapoptotic proteins, causing TRAIL-induced apoptosis in human melanoma cells (17). These findings suggested that combination therapy of anisomycin and lexatumumab could be effective in the treatment of melanoma. In the present study, we showed that anisomycin alone at similar concentrations suppressed the proliferation of B16 mouse melanoma cells.

One limitation of this study is that the effects of anisomycin on mouse melanoma cells might not necessarily reflect its effects on human melanoma cells. It has been reported that cells of rodents, such as mice and rats, have faster oscillatory dynamics of protein metabolism than that seen in human cells (41, 42), which might enhance the sensitivity of B16 mouse melanoma cells to anisomycin.

In conclusion, this study is the first to evaluate the growth-suppressing effect of anisomycin on mouse melanoma cells. The findings suggest that anisomycin might exert a growth-suppressing effect via reduction of the intracellular NAD level in B16 cells, which causes cell senescence-like phenomena and increases the expression levels of the tumor suppressor gene Txnip. These results suggest that anisomycin may hold promise as an anticancer drug that is effective at low concentrations against melanoma.

Acknowledgements

This research was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP19K11749.

Footnotes

  • Authors’ Contributions

    Hironori Ushijima: Conceptualization, Methodology, Investigation, Resources, Data curation, Validation, Formal Analysis, Visualization, Supervision, Writing, Funding acquisition, Project administration. Rina Monzaki: Methodology, Investigation, Data curation, Validation, Formal Analysis, Visualization. Arisa Onodera: Investigation, Data curation, Visualization.

  • Conflicts of Interest

    The Authors declare that there are no conflicts of interest or any competing financial interest.

  • Received August 3, 2021.
  • Revision received September 21, 2021.
  • Accepted September 22, 2021.

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Suppressive Effects of Anisomycin on the Proliferation of B16 Mouse Melanoma Cells In Vitro
HIRONORI USHIJIMA, RINA MONZAKI, ARISA ONODERA
Anticancer Research Dec 2021, 41 (12) 6113-6121; DOI: 10.21873/anticanres.15431
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