[6]-Gingerol Decreases Clonogenicity And Radioresistance of Human Prostate Cancer Cells

A B S T R A C T

The phenolic compound [6]-Gingerol, isolated from Zingiber officinale, has been demonstrated to have antitumor activity for different types of malignant tumours. Prostate cancer is the most common malignancy among males worldwide, being the second leading cause of cancer death in men. In the present study, we investigated the antitumor action of [6]-Gingerol on a human prostate cancer cell line (LNCaP). Our data shows that [6]-Gingerol treatment induced a dose-dependent decrease in the cell viability. Compared with the vehicle control, the cell viabilities were 79.90 ± 3.56% and 53.06 ± 7.82% when the LNCaP cells were exposed to 150 μg/mL and 300 μg/mL of [6]-Gingerol, respectively. The treatment of LNCaP with 300 µM of [6]-Gingerol led to a significant reduction (~25%) on the clonogenic survival of these cells. Furthermore, [6]-gingerol acted as a radiosensitizer for LNCaP cells. The pretreatment of these cells with [6]-Gingerol significantly enhanced the killing effects of ionizing radiation with a dose enhancement ratio of 1.25. Our results demonstrate the anti-tumour activities of [6]-Gingerol. Further studies are needed to elucidate the mechanisms involved.

Keywords

Prostate cancer, [6]-Gingerol, cell viability, clonogenicity, radiosensitizer

Introduction

Prostate cancer (PCa) is the second most prevalent malignancy and second leading cause of cancer-related deaths among men in the world [1]. The choice of treatment modality depends on the stage of the disease and the patient´s clinical conditions [2]. Radical prostatectomy combined with radiotherapy (RT) is standard treatment for clinically localized PCa. Unfortunately, a significant percentage of RT-treated patients develop locally persistent or recurrent tumours [3, 4].

Herbal medicine involves the treatment of disease based on the use of plants and plant extracts [5]. A large number of clinical studies have reported the therapeutic benefits of herbal medicines in combination with conventional therapeutics for the treatment of cancer patients [6-8]. Ginger (Zingiber officinale Roscoe, Zingiberaceae) is largely used in diets worldwide and also in traditional oriental medicine [9]. The compound [6]-Gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3-decanone) is the most abundant, biologically active constituent of Ginger (Figure 1) [10]. [6]-Gingerol has been reported to have anti-oxidant, anti-inflammatory, and anti-tumour activities [9, 11-13]. In the present study, we investigated the anticancer properties of [6]-Gingerol in human PCa cells.

Materials and Methods

I Materials

The follow materials were used: Roswell Park Memorial Institute-1640 (RPMI-1640) media; foetal bovine serum (FBS); penicillin and streptomycin both purchased from Life Technologies (Waltham, MA USA); CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) obtained from Promega (Madison, WI, USA); dimethyl sulfoxide (DMSO) and [6]-Gingerol purchased from Sigma Aldrich (Missouri, MO, USA); and LNCaP cells (CRL-1740™) obtained from American Type Culture Collection (ATCC®).

II Cell Culture

LNCap cells were cultured in RPMI-1640 with 10% Foetal Bovine Serum (FBS) along with 100 U/ml penicillin and streptomycin at a concentration of 300 µg/mL. The cell line was maintained at 37°C in a humidified atmosphere of 5% CO2 and were sub-cultured twice weekly.

III Cell viability (MTS)

Cell viability was assessed by using a [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)]-based assay. The assay was based on the reduction of tetrazolium salt through the mitochondrial dehydrogenase of intact cells into a purple formazan product. The cells were seeded into 96-well plates (2,5×103 cells/well), and incubated for 5-6 hr to facilitate attachment. Cells were then treated with 150 and 300µg/mL of [6]-Gingerol or vehicle alone (0.1% DMSO) in serum containing media, and incubated for 24hr at 37°C. After incubation, MTS solution was added to the plate at a final concentration of 0.5 mg/mL. The cells were incubated for 2 hr in the dark at 37°C. The resulting MTS-products were determined by measuring the absorbance at 490 nm with an ELISA reader.

IV Clonogenic assay

The clonogenic assay was performed to evaluate in vitro cell survival following treatment with [6]-Gingerol. For the colony formation assay, LNCaP cells (1×102 cells/dish) were divided into treatment groups with 300µg/mL of [6]-Gingerol and no treatment and seeded into 60 mm culture dishes, followed by incubation at 37°C. Ten days later, cell colonies were fixed and stained with methanol 20% and crystal violet 0.5% and colonies of at least 50 cells were counted.

V Radiosensitivity measurements

The LNCaP cells were seeded at a concentration of 100 – 2.400 cells/dish and were divided into two groups: cells which served as irradiated controls and cells treated with [6]-gingerol and irradiated. Cells were irradiated by a 60Co source in the range from 4 to 15 Gy, using the GammaCell 220 – Irradiation Unit of Canadian-Atomic Energy Commision Ltd. (CTR-IPEN). After 14 days of culture in normoxic conditions, cell colonies were fixed and stained with methanol 20% and crystal violet 0.5%; colonies of at least 50 cells were counted. The surviving fraction was calculated as the ratio of the plating efficiency of treated cells to the control cells. The dose enhancement ratio (DER) was calculated as the dose (Gy) that yielded a surviving fraction of 0.03 for control divided by that for the [6]-Gingerol treated cells.

VI Statistical Analysis

The results are presented as the mean ± S.E. Single comparisons of the mean values were completed via a Student`s t-test. Multiple comparisons were assessed by One-way ANOVA, followed by Bonferroni´s tests with GraphPad Prism version 6.0 software. A p-value < 0.05 was considered statistically significant.

VII Results and discussion

Radiotherapy is frequently combined with prostatectomy to treat localised tumours, but many patients present with recurrent or persistent disease [14]. One approach to improve the efficacy of RT is the use of radiosensitizers. The use of natural compounds as radiosensitizers could be a good therapeutic tool in oncology [15, 16].

In this work, we first investigated the effect of [6]-gingerol on viability of LNCaP prostate cancer cells. Our results demonstrated that [6]-Gingerol treatment induced a dose-dependent decrease in the cell viability (Figure 2). Compared with the vehicle control, the cell viabilities were 79.90 ± 3.56% and 53.06 ± 7.82% when the LNCaP cells were exposed to 150 μg/mL and 300 μg/mL of [6]-Gingerol, respectively. The inhibitory effect on cell viability was more prominent at a dose of 300 μM of [6]-Gingerol after 24-h of pre-treatment (P<0.001). A significant difference in cell viability was also observed between the cells treated with 150 μg/mL and 300 μg/mL of [6]-Gingerol (P<0.01), suggesting a possible [6]-Gingerol dose-dependent effect on LNCaP cancer cells.

We further investigated the effects of 300 μM of [6]-Gingerol treatment on the drug sensitive and radioresistance assays. The drug sensitive effect of [6]-Gingerol on LNCaP cells was determined by using a colony formation (clonogenicity survival) assay. This assay has been previously employed for the evaluation of drug sensitivity in tumour cell lines [17]. After a 10-day culture period, colonies were stained and counted (Figure 3A). The efficiency of colony formation was 75.11± 5.07% when the LNCaP cells were exposed to 300 μg/mL of [6]-Gingerol. (P<0.05) (Figure 3B). These results indicate that [6]-Gingerol decreased the ability of LNCaP cells to form and sustain cell proliferation. A similar effect of [6]-Gingerol was also observed in pancreatic cells [18].

To analyse the effectiveness of [6]-Gingerol as a radiosensitizer, LNCaP cells were treated with 300 μg/mL of [6]-Gingerol and irradiated at doses ranging from 4-15 Gy. Cells were grown for 14 days, after which the colonies were fixed and stained with crystal violet (Figure 4A). As shown in (Figure 4B), [6]-Gingerol induced a dose-dependent reduction on clonogenic survival. The results show that pretreatment with [6]-Gingerol significantly enhanced the cell killing effect of irradiation with a DER of 1.25, indicating its potential radiosensitizing effects in prostate cancer cells.

Taken together, our results indicate [6]-Gingerol has anti-tumour activities on the LNCaP cell line. Further mechanistic studies are necessary and are currently in progress.

Acknowledgments

This study was supported by the grants of FAPESP (2014/19265-7).

Conflicts of interest

The authors declare that that are no conflict of interest.

Article Info

Article Type

Research Article

Publication history

Received: Fri 20, Sep 2019
Accepted: Thu 03, Oct 2019
Published: Mon 30, Dec 2019

Copyright

© 2023 Maria Helena Bellini. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Hosting by Science Repository.
DOI: 10.31487/j.COR.2019.05.07

Author Info

Maria Helena Bellini Silva JPL Teixeira LF

Corresponding Author

Maria Helena Bellini
Department of Biotechnology, IPEN-CNEN/SP, São Paulo, Brazil

Figures & Tables

Figure 1: Chemical structure of the main active compound present in ginger extract ([6]-Gingerol) [10].

Figure 2: [6]-Gingerol treatment induced a decrease in the viability of LNCaP cells. Concentrations of 150 μg/mL and 300 μg/mL doses demonstrated significant reduction in cell counts ***P<0.001 vs control. Moreover, a significant difference in cell viability between the cells treated with 150 μg/mL and 300 μg/mL of [6]-Gingerol (**P<0.01). Each bar represents means ± SE, n=6. Statistical analysis was performed using One-way ANOVA followed by Bonferroni's test.

Figure 3: [6]-Gingerol decreased the clonogenic capacity of LNCaP cells. (A) Representative images of stained colonies (B) Quantitative analysis revealed a significant difference between control and the treated group (*P<0.05). Each bar represents means ± SE, n=3. Statistical analysis was performed using Student’s T test.

Figure 4: Radiosensitization of LNCaP cells. (A) Representative images of stained colonies. (B) Radiation cell survival curves of LNCaP cells with and without 300 μg/mL [6]-Gingerol treatment. Clonogenic assays show that sensitizes [6]-Gingerol to irradiation-induced cell killing. (**P<0.01, ***P<0.001) Each bar represents means ± SE, n=3. Statistical analysis was performed using One-way ANOVA followed by Bonferroni's test.

References

1. Rawla P (2019) Epidemiology of Prostate Cancer. World J Oncol 10: 63-89. [Crossref]
2. Pendleton J, Pisters LL, Nakamura K, Anai S, Rosser CJ (2007) Neoadjuvant therapy before radical prostatectomy: where have we been? Where are we going? Urol Oncol 25: 11-18. [Crossref]
3. Palacios DA, Miyake M, Rosser CJ (2013) Radiosensitization in prostate cancer: mechanisms and targets. BMC Urol 26: 4. [Crossref]
4. Datta NR, Rogers S, Ordóñez SG, Puric E, Bodis S (2016) Hyperthermia and radiotherapy in the management of head and neck cancers: A systematic review and meta-analysis. Int J Hyperthermia 32: 31-40. [Crossref]
5. Nyaba ZN, Murambiwa P, Opoku AR, Mukaratirwa S, Shode FO et al. (2018) Isolation, characterization, and biological evaluation of a potent anti-malarial drimane sesquiterpene from Warburgia salutaris stem bark. Malar J 17: 296. [Crossref]
6. Moreira DL, Teixeira SS, Monteiro MHD, De-Oliveira ACAX, Paumgartten FJR (2014) Traditional use and safety of herbal medicines. Rev Bras Farmacogn 24: 248-257.
7. Firenzuoli F, Gori L (2007) Herbal medicine today: clinical and research issues. Eur Med J Urol 1: 70-73. [Crossref]
8. Olaku O, White JD (2011) Herbal therapy use by cancer patients: a literature review on case reports. Eur J Cancer 47: 508-514. [Crossref]
9. Park YJ, Wen J, Bang S, Park SW, Song SY (2006) [6]-Gingerol induces cell cycle arrest and cell death of mutant p53-expressing pancreatic cancer cells. Yonsei Med J 47: 688-697. [Crossref]
10. Kim HW, Oh DH, Jung C, Kwon DD, Lim YC (2011) Apoptotic Effects of 6-Gingerol in LNCaP Human Prostate Cancer Cells. Soonchunhyang Med Sci 17: 75-79.
11. Lv L, Chen H, Soroka D, Chen X, Leung T et al. (2012) 6-gingerdiols as the major metabolites of 6-gingerol in cancer cells and in mice and their cytotoxic effects on human cancer cells. J Agric Food Chem 60: 11372-11377. [Crossref]
12. Poltronieri J, Becceneri AB, Fuzer AM, Filho JC, Martin AC et al. (2014) [6]-gingerol as a cancer chemopreventive agent: a review of its activity on different steps of the metastatic process. Mini Rev Med Chem 14: 313-321. [Crossref]
13. Kumara M, Shylajab MR, Nazeemc PA, Babu T (2017) 6-Gingerol is the most Potent Anticancerous Compound in Ginger(Zingiber officinale Rosc.) J Dev Drugs 6: 1.
14. Gay HA, Michalski JM (2018) Radiation Therapy for Prostate Cancer. Mo Med 115: 146-150.
15. Linam J, Yang LX (2015) Recent developments in radiosensitization. Anticancer Res 35: 2479-2485. [Crossref]
16. Malik A, Sultana M, Qazi A, Qazi MH, Parveen G et al. (2016) Role of Natural Radiosensitizers and Cancer Cell Radioresistance: An Update. Anal Cell Pathol 2016: 6146595. [Crossref]
17. Kawada K, Yonei T, Ueoka H, Kiura K, Tabata M et al. (2002) Comparison of chemosensitivity tests: clonogenic assay versus MTT assay. Acta Med Okayama 56: 12ss9-134. [Crossref]
18. Park YJ, Wen J, Bang S, Park SW, Song SY (2006) [6]-Gingerol induces cell cycle arrest and cell death of mutant p53-expressing pancreatic cancer cells. Yonsei Med J 47: 688-697. [Crossref]