Ponicidin Induces Apoptosis of Murine Melanoma by Inhibiting the NF-κB Signaling Pathway

Abstract

Ponicidin (PON), a diterpenoid extracted from the Chinese herb Rubescens, has been reported to be a therapeutic cytotoxic drug for the treatment of various types of human cancers. According to the statistics, the incidence of malignant melanoma is increasing year by year and the degree of malignancy is extremely high, so early treatment is very important. In the present study, we demonstrated the antitumor effect of PON on melanoma in vitro and in vivo. Cell Counting Kit-8 (CCK-8) assay was used to detect cell proliferation rate, crystal violet staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) kit was used to detect cell apoptosis, and Western blotting was used to detect the expression of apoptotic indicators and related signaling pathway proteins. Finally, the tumor-bearing mouse model was constructed. Treating melanoma B16F0 and B16F10 cells with different concentrations (10 and 20 µmol/L) of PON magnificantly decreased cell viability. In addition, PON significantly activates the expression of pro-apoptotic proteins, including cleaved-poly(ADP-ribose)polymerase (PARP) (cl.PARP), Bak and Bim proteins, and also inhibits the expression of anti-apoptotic protein Mcl-1 and nuclear transcription factor nuclear factor-kappaB (NF-κB) in melanoma cells. Lastly, PON effectively inhibits the growth of mouse xenografts in vivo. These results suggest that PON induces apoptosis of melanoma cells may be achieved by inhibiting NF-κB signal pathway, but the specific mechanism remains to be further elucidated. Taken together, PON may serve as an effective potential drug for the treatment of melanoma.

INTRODUCTION

Melanoma is a malignant tumor derived from melanocytes that occurs on the surface of the skin.¹⁾ It is the most serious and deadly type of skin cancer, accounting for 6.8 to 20% of skin cancers.²⁾ Epidemiological studies have shown that the number of melanoma patients in the world has increased year by year in recent years, and the mortality rate of patients is relatively high, which poses a serious threat to people's life and health.³⁾ Although many scholars have conducted extensive research on melanoma and improved treatment options,^4,5) it remains a great challenge for clinicians to completely cure melanoma. The main characteristics of melanoma are poor prognosis, high mortality, and rapid proliferation. Controlling its proliferation has become the key to curing melanoma. At present, there are some chemotherapeutic drugs used for the prevention and treatment of melanoma in clinical practice,^6–9) but the effect is not very satisfactory, and most patients have to face the problem of drug resistance after receiving chemotherapy. The current clinical treatments for melanoma are immunotherapy, targeted therapy and surgical resection. However, there are problems such as large trauma, obvious side effects, and short effective treatment time. Therefore, finding safe and effective drugs for melanoma therapy has become an urgent problem to be solved.

Rabdosia rubescens is an edible traditional Chinese herbal medicine for esophageal cancer and gastric cancer. The main active component of Rabdosia rubescens is Ponicidin (PON)^10–12) (Fig. 1), which is an ent-kaurane diterpenoid. PON has been reported to have immunomodulatory, anti-inflammatory and anti-tumor effects on various cancer types.¹³⁾ Recent studies have shown that PON can induce apoptosis in human breast cancer cells,¹⁴⁾ human gastric cancer cells,¹⁵⁾ and lung cancer cells.¹⁶⁾ In addition, it also inhibits the epithelial-mesenchymal transition and metastasis of colorectal cancer cells.¹⁷⁾ Furthermore, they can also improve the efficacy when combined with other chemotherapy drugs,¹⁸⁾ and reduce drug toxicity and side effects. Although a large number of experimental studies have shown that PON has an inhibitory effect on a variety of different tumor cells,^15,19–22) the effects of PON on melanoma cells and its underlying mechanism remains unclear.

Fig. 1. Chemical Structure of Ponicidin (PON)

Nuclear factor-kappaB (NF-κB) is a nuclear transcription factor that can regulate the transcription process of multiple genes in response to various extracellular signaling stimuli, thus affecting cell proliferation.^23,24) Previous studies have shown that the NF-κB signaling pathway plays an important role in the survival of melanoma cells, and the activation of the NF-κB transcription factor is an important pathway for melanoma to achieve survival, proliferation, and resistance to apoptosis.^25,26) However, the inhibitory effect of PON on NF-κB signaling pathway in melanoma remains unclear. Therefore, in this study, we detected the p65 protein of NF-κB (composed of two subunits p65 and p50).

The purpose of this study was to evaluate the anti-tumor effect of PON on melanoma. In vitro and in vivo experiments, we found that PON can effectively induce apoptosis of murine melanoma B16F10 and B16F0 cells, and this may be achieved by inhibiting NF-κB signal pathway. In conclusion, PON might be used as a potent candidate for the development of new drug for the treatment of melanoma.

MATERIALS AND METHODS

Reagents

Ponicidin was purchased from MedChemExpress (Monmouth Junction, NJ, U.S.A.) and purchased from Chengdu Purechem-Standard Co., Ltd. (Sichuan, China). Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and IKK 16 (HY-13687) were purchased from MedChemExpress. Cell Counting Kit-8 (CCK-8) was purchased from Yeasen Biotechnology (Shanghai) Co., Ltd. (Shanghai, China). The antibody against cleaved-poly(ADP-ribose)polymerase 1 (PARP1) (ab32064) was purchased from Abcam (Cambridge, MA, U.S.A.). Antibodies against Bak (#12105), Bad (#9239), Bim (#2933), Mcl-1 (#5453), Bcl-xL (#2764), Bcl-2 (#3498), Phospho-NF-κB p65 (Ser536) (#3033) and NF-κB p65 (#8242) were purchased from Cell Signaling Technology, Inc. (Beverley, MA, U.S.A.). Antibody against Cdk4 (DCS-35): sc-23896 and cyclin D3 (C-16): sc-182 were purchased from Santa Cruz Biotechnology (Shanghai) Co., Ltd. (Shanghai, China). Antibody against β-actin was purchased from Proteintech (Wuhan, China). The secondary antibodies were supplied by Cell Signaling Technology and Abways Technology (Shanghai, China), respectively.

Cell Culture

The cell lines used in the experiments were murine B16F10 and B16F0 melanoma cells. These cell lines were cultured in complete medium containing 1% streptomycin/penicillin and 10% fetal bovine serum (FBS). Cells were cultured at 37 °C in an incubator containing 5% CO₂ and the medium was changed once a day.

Cell Viability

Melanoma B16F10 and B16F0 cells in logarithmic growth phase were seeded in 96-well plates with 10⁴ cell per well. After the cells adhered, the drug group was treated with different concentrations of PON (0, 10, 20 µmol/L). The control group was added an equal volume of carrier (0.2% dimethyl sulfoxide). After incubation for 24 h, 10 µL of CCK-8 solution was added to each well, and the culture plate was placed in an incubator for incubation for 1–4 h. Absorbance was measured at 450 nm wavelength with a microplate reader.

Crystal Violet Staining

B16F0 and B16F10 cells in logarithmic growth phase were digested with trypsin, the concentration of cells was adjusted to 1 × 10⁵/mL in medium after centrifugation, and the cells were inoculated in 24-well plates. On the second day, when the cells grew to about 90%, PON with different concentrations (0, 5, 10, and 20 µmol/L) was prepared in serum-free medium. The cells were cultured for 24 h, the supernatant was discarded, washed with phosphate buffered saline (PBS) for three times, fixed with 4% paraformaldehyde at room temperature for 15 min, and stained with 1% crystal violet solution for 25 min. Finally, each group was photographed with a camera.

Tdt-Mediated dUTP Nick-End Labeling (TUNEL) Analysis

B16F0 cells at the logarithmic growth stage were inoculated in 24-well plates and cultured overnight at 37 °C for 24 h, then treated with different concentrations of PON for 24 h. Then wash with PBS for three times and fix with 4% paraformaldehyde. After fixation, place at 4 °C for 25 min, incubate with Proteinase K for five minutes at room temperature for permeabilization, and then add the diluted TdT solution in the dark, mix well and add it to each group to ensure that the liquid surface covers the bottom of the 24-well plate. Fluorescence microscope was used for observation, and the staining and apoptosis were analyzed. The methods of cell plating and drug administration were the same as above.

Western Blot

Cells were washed three times with pre-cooled PBS, an appropriate amount of radio immunoprecipitation assay (RIPA) lysis buffer was added to each well, and the cells were kept on ice for five minutes. The cells were scraped from the bottom with a cell scraper and placed in a centrifuge tube. Vortexed for 30 s and then let stand for five minutes, this step was repeated three times. Turn on the 4 °C centrifuge in advance, and centrifuge at 15000 × g for ten minutes after standing to remove the precipitate. The protein concentration was measured using the Bradford assay. Find the corresponding loading amount on the standard curve, use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technology to separate the protein, and then transfer the protein on the gel to polyvinylidene fluoride (PVDF). The PVDF membrane was soaked in 5% skimmed milk prepared in advance with TBST and then placed on a shaker for two hours at room temperature. PVDF membranes were incubated with primary antibodies to cleaved-PARP (cl.PARP), Bak, Bim, Mcl-1, β-actin, p65 and p-p65 and placed at 4 °C. The primary antibody was recovered the next day, the PVDF membrane was washed three times with TBST, the corresponding secondary antibody was incubated at room temperature, and the PVDF membrane was washed three times after one hour. Finally, bands were imaged with the Amersham Image 600 and grayscale analysis by ImageJ.

Tumor Growth in Vivo and Drug Treatment

A total of 30 female C57BL/6 mice 6–8 weeks old (about 20 g) were used in this study. These mice were purchased from Shan-dong Provincial Laboratory Animal Center. All animal experiments were approved by the Ethics Committee of Xinxiang Medical University.

Mice used in this study were maintained on a dark/light cycle of 12 h duration with food and water provided ad libitum. B16F10 cells (10⁶) in logarithmic growth phase were inoculated subcutaneously in the right forelimb of mice. Thirty tumor-bearing mice were randomized into three groups when the mean tumor volume reached 80–100 mm³: PON low-dose group (10 mg/kg); PON high-dose group (20 mg/kg); model control group was given an equal volume of normal saline. Animals in each group were intraperitoneally administered for nine consecutive days, once a day. Tumor volumes was measured by once every two days and calculated according to the formula 1/2 × length × width², and the tumor growth curves of each group were drawn and compared. At the termination of the experiment, mice were euthanized, and the complete subcutaneous tumor was quickly removed, weighed, and the tumor inhibition rate of each group was calculated and analyzed. Afterwards, the tumor inhibition rate RI% = (m⁰ − mⁿ)/m⁰ × 100% was calculated (m⁰ is the tumor weight in the model group, and mⁿ is the tumor weight in the treatment group). Finally, some tumor tissues were stored in −80 °C refrigerator for molecular detection, and some were fixed with 4% paraformaldehyde and then paraffin sections were prepared and immunofluorescence staining tests were performed with TUNEL staining.

Statistical Analysis

Study data were collected in standard forms and checked for completeness. GraphPad Prism 7.0 software was used for statistical analysis of the data. The measurement data conforming to the normal distribution were described in the form of mean ± standard deviation (S.D.), and the comparison between groups was performed by one-way ANOVA followed by Tukey’s test. A p value of less than 0.05 (2-sided significance testing) was considered statistically significant in all analyses. All experiments were performed in at least triplicate.

RESULTS

PON Induces Cell Death in Murine Menlanoma B16F10 and B16F0 Cells

To determine the effects of PON on melanoma cells, murine melanoma B16F10 and B16F0 cells were treated with different concentrations of PON (10 and 20 µmol/L) for 24 h, and cell viability was detected by CCK-8 assay. As a result, PON had a significant growth inhibition effect on B16F10 and B16F0 cells (Fig. 2a). To further verify the effect of PON, B16F10 and B16F0 cells were seeded in a 24-well plate and treated with different concentrations of PON. After incubation for 24 h, the cells were stained with 0.1% crystal violet. As shown in Fig. 2b, after the addition of PON, the cell viability was significantly reduced.

Fig. 2. PON Decreases the Viability of B16F10 and B16F0 Cells

(a) Cell Counting Kit-8 (CCK-8) assay was performed. Compared with the control group (ctl), PON (10 and 20 µmol/L) significantly inhibited B16F10 and B16F0 cells viability. (b) The effects of different concentrations of PON (0, 5, 10, and 20 µmol/L) on B16F10 and B16F0 cells were examined by crystal violet staining. The data are presented as the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01, as compared to the control group.

PON Induces Apoptosis of B16F10 and B16F0 Cells by Regulating the Expression of bcl-2 Family Proteins

To elucidate how PON induces melanoma cell death, expression of apoptosis-related proteins was detected by Western blotting. As shown in Fig. 3, after treating B16F10 and B16F0 cells with PON for 24 h, cl.PARP1, Bak, Bim protein levels increased in a dose-dependent manner, while Bad protein levels did not change significantly. Compared with the control group, PON treatment for 24 h also obviously decreased the protein level of Mcl-1, but had no effect on the protein levels of Bcl-2 and Bcl-xl (Fig. 4). To further verify that PON inhibited the growth of melanoma cells through apoptosis, we performed TUNEL assay. We found that the fluorescence of B16F10 cells increased after PON treatment (Fig. 5). These results indicate that PON treatment induced cell death of melanoma cells through apoptosis.

Fig. 3. PON Increases the Expression of Pro-apoptotic Proteins in B16F10 and B16F0 Cells

(a) and (e) B16F10 and B16F0 cells were treated with PON (0, 10, and 20 µmol/L) for 24 h, and apoptosis-related proteins was detected by Western blotting and (b–d) and (f–h) quantitative analyses. β-Actin was used as a loading control. Results showed that cleaved-PARP (cl.PARP), Bak and Bim were up-regulated. The Bim antibody used in this study can detect endogenous levels of total Bim detected. The data are presented as the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, as compared to the control group.

Fig. 4. PON Decreases the Expression of Anti-apoptotic Proteins in B16F10 and B16F0 Cells

(a) and (c) B16F10 and B16F0 cells were treated with different concentrations of PON for 24 h, and the expression of apoptosis-related proteins was detected by Western blotting and (b) and (d) quantitative analyses. β-Actin was used as a loading control. Results showed that Mcl-1 was down-regulated significantly. The data are presented as the mean ± S.D. (n = 3). * p < 0.05, *** p < 0.001, as compared to the control group.

Fig. 5. PON Induces Apoptosis of B16F10 Cells

(a) TUNEL staining of melanoma B16F10 cells after PON treatment for 24 h. Apoptotic cells are red and nuclei are stained blue with DAPI (×100). Scale bar: 200 µm. (b) The average number of apoptotic cells in each treatment group. The data are presented as the mean ± S.D. (n = 3). * p < 0.05, *** p < 0.001, as compared to the control group.

PON Induces Apoptosis of B16F10 and B16F0 Cells through NF-κB Signaling Pathway

In order to study the signaling pathways involved in PON-induced apoptosis, we detected the expression of apoptosis-related signaling pathways, including p65 and p-p65 by Western blotting. We found that p65 phosphorylation levels were significantly reduced in B16F10 and B16F0 cells after PON treatment compared to the control group (Figs. 6a–d). To clarify the contribution of NF-κB signaling pathway in PON-induced apoptotic cell death of murine melanoma B16F10 and B16F0 cells, we treated B16F10 and B16F0 cells with 10 µmol/L IKK 16 (an IKK inhibitor) for 4 h (Fig. 6e). We found that the expression of cl.PARP was significantly increased after NF-κB inhibition. To further confirm the involvement of NF-κB in PON-mediated toxic effect on B16F10 cell, we pretreated B16F10 cell with NF-κB activator (10 µmol/L PMA) for one hour, before treating B16F10 cell with PON. We found that when NF-κB was activated, the expression of cl.PARP protein was significantly decreased (Fig. 6f). These results indicate that PON induces apoptosis of B16F10 and B16F0 cells through NF-κB signaling.

Fig. 6. PON Inhibits the NF-κB Signaling Pathway in B16F10 and B16F0 Cells

(a) and (c) B16F10 and B16F0 cells were treated with different concentrations of PON for two hours, and the expression of p65 (NF-κB) was detected by Western blotting and (b) and (d) quantitative analyses. (e) Role of NF-κB signaling pathway in PON-induced apoptosis of mouse melanoma B16F10 and B16F0 cells. Western blotting results showed that phosphorylated NF-κB was inhibited, and cl.PARP protein expression was increased. (f) NF-κB is involved in PON-mediated cytotoxicity of B16F10. The expression of cl.PARP protein was detected by Western blotting. The expression of phosphorylated NF-κB increased and the expression of cl.PARP decreased. β-Actin was used as a loading control. The results showed that the phosphorylation level of p65 was significantly inhibited after PON treatment. The data are presented as the mean ± S.D. (n = 3). *** p < 0.001, as compared to the control group.

PON Suppresses the Growth of Melanoma in Mice

To further confirm the roles of PON on the growth of melanoma cells in vivo, B16F10 cells were subcutaneously transplanted into mice. After four days, when small tumor nodes were detected, different concentrations of PON were injected once a day, and the model control group was injected with the same volume of normal saline. Further analysis of the tumor growth curve showed that the growth rate of tumor volume decreased after administration of PON, and the tumor volume was significantly smaller than that of the model control group after administration of PON, especially the high-dose PON group (Figs. 7a, b). As shown in Fig. 7d, there was little difference in the body weight of the three groups of tumor-bearing mice before and after treatment, and the difference was not statistically significant (p > 0.05). By measuring the weight of the melanoma, it was found that the tumor weight of the two treatment groups was significantly smaller than that of the model group (p < 0.05) (Fig. 7c), suggesting that PON indeed retarded not only in vitro but also in vivo growth of melanoma cells. According to the calculation, the tumor inhibition rates (RI) of the low-dose and high-dose groups were 40.64 and 63.43%, respectively. As shown in Figs. 7e and f, we detected the protein expression levels of cl.PARP in each group of tumor tissues and found that the protein expression showed the same trend as that at the cellular level. Compared with the model control group, the expression of cl.PARP in tumor tissue increased in all administration groups, and the inhibition was strongest in PON high-dose group. To further confirm the effect of PON on melanoma cells, TUNEL assay was performed on tumor sections. As expected, compared with the control group and the low-dose group, the number of apoptotic cells in the tumor tissue of the high-dose group increased. All these data indicate that PON can induce the apoptosis of subcutaneous tumor tissue of melanoma cells in mice (Fig. 7g).

Fig. 7. PON Suppresses the Growth of Melanoma in Vivo

(a) B16F10 cells melanoma specimen. (b) Tumor growth curve. Tumor volume was measured and calculated after treatment. (c) Effect of PON on the weight of transplanted tumor in mice. (d) Body weights of mice before and after administration. (e) Effect of PON on cl.PARP protein expression in B16F10 subcutaneous cell graft tumor and (f) quantitative analyses. (g) Apoptosis was detected by TUNEL staining and the average number of apoptotic cells in each treatment group. Scale bar: 20 µm. The data are presented as the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, as compared to the control group.

DISCUSSION

Melanoma is a highly malignant tumor caused by abnormal melanocyte proliferation, which has high mortality and poor prognosis. PON is reported to have anti-inflammatory and anticancer properties. Therefore, the purpose of this experiment is to study whether PON has anti-melanoma effect and its potential mechanism of action.

PON is extracted from the traditional Chinese medicine Rubescens,^27,28) which has strong anti-inflammatory, antioxidant and anti-tumor effects. As a natural ingredient, PON has the advantages of low toxicity, small adverse reactions and low cost, and has a good clinical application prospect. Cui et al.²⁹⁾ found that PON can inhibit the growth of pancreatic cancer by inducing ferroptosis. The difference from our study is that PON in this report induces pancreatic cancer cell death via ferroptosis, however, our study is that PON induces melanoma cell death via apoptosis. Liu et al.³⁰⁾ found that PON can induce apoptosis of leukemia cells by activating caspase-3 in vitro. Zhang et al.²¹⁾ reported that PON induces apoptosis in hepatoma cells QGY-7701 and HepG-2 by up-regulating the expression of Bax protein and down-regulating the expression of Survivin and Bcl-2 proteins. The above reports are consistent with our research results that PON induces apoptosis by regulating the expression of Bcl-2 family proteins, thereby achieving anti-cancer effects. For example, we demonstrated that PON induced apoptosis by up-regulating the expression of Bak and Bim proteins and down-regulating the expression of Mcl-1 proteins. Later, the potential mechanism of PON induced apoptosis of melanoma cells was also investigated.

In this study, the melanoma B16F10 and B16F0 cells treated with PON could significantly inhibit cell viability. It is a remarkable fact that TUNEL staining and crystal violet staining also confirmed that PON can induce melanoma cell apoptosis in vitro. Surprisingly, PON-induced apoptosis of melanoma cells is likely to be achieved through inhibition of NF-κB. In vivo experiments, we found that PON can significantly inhibit the growth of tumor cells in the B16F10 mouse xenograft model. Not only that, we observed and analyzed almost no change in the body weight of the mice during the treatment period. In addition, PON can induce the expression of proapoptotic protein cl.PARP, which is consistent with the results of in vitro experiment. In addition, TUNEL staining experiments in tumor tissues show that PON can indeed induce apoptosis in tumor tissues.

As far as we know, this is the first report on the effect and mechanism of PON in inducing apoptosis in melanoma cells B16F10 and B16F0 in vitro and in vivo. Therefore, PON may be a safe and effective anticancer drug that induces melanoma cell apoptosis.

CONCLUSION

Collectively, it is proved for the first time that PON can significantly induce the apoptosis of melanoma cells B16F10 and B16F0 by inhibiting the NF-κB signaling pathway. In addition, the results of PON-induced apoptosis in melanoma cells were also validated in vivo. This study provides a strong theoretical basis for the application of PON as a candidate drug for the treatment of melanoma. Although the specific molecular mechanism by which PON induces melanoma cell apoptosis remains to be further elucidated, we will try our best to address this issue in the future. These results lay a solid foundation for further research on the molecular mechanism of PON against melanoma.

Acknowledgments

This study was supported by the Science and Technology Innovation Team Program of Henan University (22IRTSTHN030) and the Natural Science Foundation of Henan Province (212300410382).

Conflict of Interest

The authors declare no conflict of interest.

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