Anti-inflammatory effect of Varthemia iphionoides extracts against prostate cancer in vitro

Abstract

Varthemia iphionoides is a Jordanian medicinal plant with several health-promoting properties, including antibacterial, antioxidant and anticancer activities. However, its anti-inflammatory properties have been poorly investigated up to date. The current study aimed to investigate the anti-inflammatory effect of V. iphionoides by measuring the production of interleukin-6 in response to a pro-inflammatory stimulus (bacterial lipopolysaccharide) in in vitro cell models of human MRC-5 and PC3 cells. We observed a significant reduction in lipopolysaccharide-induced interleukin-6 release in response to V. iphionoides (125 µg/mL) in both non-cancerous fibroblast MRC-5 and prostate cancerous PC3 cells. However, the anti-inflammatory effect of this medicinal plant was stronger when MRC-5 cells were treated with an aqueous extract, while the methanolic extract was more potent in PC3 cells. The effect of V. iphionoides in reducing interleukin-6 production was not due to its cytotoxicity, and future studies are required to elucidate the mechanisms of action by which this medicinal plant modulates inflammatory responses. In conclusion, the results of our study represent the first report of the potential protective effect of water and methanolic extracts of V. iphionoides against pro-inflammatory stimuli in fibroblasts and cancer cells of human origin, and it is critically important to identify the phytochemical compounds responsible for this effect.

Introduction

Varthemia iphionoides (V. iphionoides) is one of the important medicinal herbs found in the eastern Mediterranean region. It belongs to the Asteraceae family, ¹ and it is a 30- to 80-cm-long evergreen-dwarf shrub with leaves covered with hairs and aromatic glands.

This plant has tubular flowers, and the flowering season extends from September to December. ² It has been used in traditional medicine to relieve pains, clear flatulence, heal wounds, and to treat eye complaints and urine retention. ³ It has also been shown to exhibit antimicrobial,^4,5 antiplatelet, ⁶ and cytotoxic activities. ⁷ Recently, it has been found that V. iphionoides has antidiabetic^8,9 and anticancer properties.^10,11 However, there is little evidence on its potential anti-inflammatory effect. ¹² It is well established that inflammation is associated with the progression of cancer including prostate cancer. In Jordan, prostate cancer is third only to colon and lung cancer as the leading cause of cancer-related deaths in men. ¹³ Chronic inflammation is a condition where the production rate of pro-inflammatory cytokines is accelerated, and there is a persistent activation of the immune response. ¹⁴ Continual activation of immune system can lead to malfunction of tissues and has been associated with cancer development. ¹⁵ There is strong evidence that chronic inflammation plays a key role in the development and progression of prostate cancer.^16–18

It has been suggested that one of the important strategies for future anti-inflammatory therapies is to target fibroblasts due to their important role in the inflammatory responses and chronic inflammation; they produce extracellular matrix proteins, which can influence the shape, movement and the state of activation of inflammatory cells in the tissue, as well as playing an important role in recruiting inflammatory cells to certain sites and controlling a number of cytokines.^19,20 To the best of our knowledge, no study has investigated the activity of water and methanolic extracts of V. iphionoides in fibroblasts. Therefore, the current study aimed to investigate the anti-inflammatory activity of water and methanolic extracts of V. iphionoides in human fibroblasts, and how its potential anti-inflammatory effect could be protective against prostate cancer. Since inflammation has been associated with cancer progression, notably prostate cancer, we investigated the anti-inflammatory effect of the different extracts of V. iphionoides in prostate cancer. It has been found that the pro-inflammatory interleukin-6 (IL-6) is associated with prostate cancer progression,^15,21,22 and could represent a potential target for cancer therapies. We explored the effect of both aqueous and organic extracts of V. iphionoides on IL-6 production in a human prostate cancerous cell line, PC3, which was chosen as in vitro model of prostate cancer. This is the first study to investigate the effect of water and methanolic extracts of V. iphionoides on the secretion of inflammatory mediators in non-cancerous and cancerous human cells, and has provided new insights on the biological profile of this promising Jordanian medicinal plant.

Materials and methods

Chemical reagents and solutions

Bacterial lipopolysaccharides (LPS) were purchased from Sigma-Aldrich (United States) and reconstituted in di-ionised water to prepare 5 mg/mL stock solution. Dimethyl sulfoxide (DMSO) stock solution was purchased form Duchefa (Netherland). All stock solutions were diluted to the required final concentrations using media supplemented with 10% heat-inactivated foetal bovine serum (FBS). Water soluble tetrazolium (WST)-1 proliferation reagent was purchased from Abcam (United Kingdom).

Preparation of medicinal plant extracts

Water and methanolic extracts were prepared from the leaves of V. iphionoides. The leaves were separated from the plant and washed with distilled water, dried at room temperature and then grinded using a blender. The resulting plant powder (10 g) was extracted by maceration with 100 mL of water or methanol for 72 h at room temperature with shaking at 150 rpm. All samples were filtered through white cheese cloth paper and Whatman filter papers of size 11.00 cm. Solvents were evaporated by rotary evaporator and water bath at 45°C. The crude extracts were dissolved in DMSO to a final stock concentration of 10 mg/mL. All extracts were purified by filtration through 0.22 µm filter units and kept at −20°C.

Cell cultures

Non-cancerous human lung fibroblasts (MRC-5) (ATCC # CCL171) and human prostate adenocarcinoma (PC3) (CLS # CVCL0035) cells were a gift from Dr. Malek Zihlif and Dr. Mamoun Ahram (Faculty of Medicine, University of Jordan, Jordan) and Ms. Bashaer Abu-Irmaileh (Hamdi Mango Research Centre, Jordan). Cells were routinely cultured in RPMI 1640 medium supplemented with 100 mmol/L glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin and 10% heat-inactivated FBS (United States). Both cell lines were grown to 70% confluence at 37°C in a humidified atmosphere of 5% CO₂.

WST-1 viability assay

Cell viability in response to water and methanolic extracts of V. iphionoides was determined using WST-1 viability assay. MRC-5 and PC3 cells were seeded in 96-well culture plates in a final volume of 100 mL/well culture medium and cultured in a humidified atmosphere (37°C, 5% CO₂). Cells were allowed to adhere to the plate surface for 36 h before being treated with V. iphionoides (0–400 µg/mL) or vehicle control (DMSO) for 72 h. WST-1 reagent (10 mL) was added to each well and incubated for 30 min in a humidified atmosphere (37°C, 5% CO₂). Quantification of the formazan dye produced by metabolically active cells was performed by a scanning multiwell spectrophotometer, measuring absorbance at 570 nm with a reference wavelength of 630 nm by a microplate reader (TECAN, Austria).

Measuring IL-6 protein secretion

MRC-5 and PC3 cells were allowed to adhere to the plate surface for 36 h before being treated with plant extracts (15.6 and 125 µg/mL water or methanolic extracts) or vehicle control (DMSO) for 72 h in a humidified atmosphere (37°C, 5% CO₂). Cells were then exposed to a pro-inflammatory bacterial agent, LPS, by replacing culture media with fresh media containing LPS at the concentration of 500 mg/mL and plant extracts (15.6 and 125 µg/mL water or methanolic extracts). DMSO was used as vehicle control. Cells were exposed to LPS in the presence of the plant extract for 24 h. Supernatants were then collected and appropriately stored at −20°C until analysis. IL-6 levels in supernatants were quantified by using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Sigma-Aldrich) following manufacturer’s instructions.

Statistical analysis

Data were expressed as mean ± standard deviation (SD). Outlier removal and check for normality of residuals were performed before statistical comparisons of the results, made using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-test. The statistical analysis was performed using GraphPad Prism Software (GraphPad Software Inc., San Diego, CA, USA).

Results

Effects of V. iphionoides on the pro-inflammatory cytokine IL-6

To assess the anti-inflammatory effects of V. iphionoides water and methanolic extracts, IL-6-induced LPS secretion was measured in cells pre-treated with plant extracts at the final concentrations of 15.6 and 125 µg/mL for 72 h. The bacterial pro-inflammatory agent LPS was able to significantly induce IL-6 production in MRC-5 (P < 0.001). The pre-treatment with 125 µg/mL V. iphionoides water extract significantly reduced IL-6 secretion in response to LPS compared to the LPS-treated cells (P < 0.05) (Figure 1(a)).

Figure 1.

Effect of Varthemia iphionoides extracts on IL-6 in MRC-5 cells. IL-6 secretion in response to pre-treatment with V. iphionoides (a) water and (b) methanolic extracts in lipopolysaccharide (LPS) (500 ng/mL)-treated fibroblastic cells. Interleukin-6 (IL-6) was measured in culture medium using a commercially available human IL-6 ELISA kit. Results represent means ± SD of three biological replicates. Statistical analysis was performed using a one way ANOVA followed by Tukey’s multiple comparison post-test (***P < 0.001, **P < 0.01, *P ⩽ 0.05).

We observed a similar response following treatment with the methanolic extract; however, the effect induced by V. iphionoides methanolic extract was not statistically significant (Figure 1(b)).

In PC3 cells, V. iphionoides water extract did not change the levels of IL-6 induced by challenging the cells with the pro-inflammatory agent LPS (Figure 2(a)).

Figure 2.

Effect of Varthemia iphionoides extracts on IL-6 in PC3 cells. IL-6 secretion in response to pre-treatment with V. iphionoides (a) water and (b) methanolic extracts in lipopolysaccharide (LPS) (500 ng/mL)-treated PC3 cells. Interleukin-6 (IL-6) secretion was measured in culture medium using human IL-6 ELISA kit. Results represent means ± SD of three biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison post-test (*P ⩽ 0.05).

In contrast, we observed an effect of the methanolic extract at the concentration of 125 µg/mL in reducing LPS-induced IL-6 secretion compared to LPS-treated cells (P ⩽ 0.05) (Figure 2(b)).

Cell viability in response to V. iphionoides in MRC-5 and PC3 cells

Results obtained from the WST-1 assay show that in MRC-5 cells treatment with V. iphionoides (0–400 µg/mL) water and methanolic extracts was not associated with any toxic effect (P ⩽ 0.05) (Figure 3(a)).

Figure 3.

Cell viability in response to Varthemia iphionoides water and methanolic extracts. Viability assay carried out in (a) MRC-5 and (b) PC3 cells in response to V. iphionoides water (closed circle; ●) and methanolic (closed square; ■) extracts (0–400 µg/mL) for 72 h. Results represent means ± SD of three biological replicates. In MRC-5 cells, the treatment with water extract significantly induced cell growth compared to the control except for 12.5 and 100 µg/mL; each concentration of the methanolic extract significantly increased cell growth compared to the control. In PC3 cells, plant extracts did not significantly change the cells’ growth, except 200 and 400 µg/mL; Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison post-test (*P ⩽ 0.05, ***P ⩽ 0.001).

In contrast, V. iphionoides methanolic extract significantly induced cell growth at all concentrations (P ⩽ 0.05). A similar effect was observed in MRC-5 treated with the water extract except at the concentrations of 12.5 µg/mL and 100 µg/mL. No changes in cell viability were observed by exposing PC3 cells to a wide range of concentration of the plant extracts (0–400 µg/mL) (Figure 3(b)) (P ⩽ 0.05). This indicates that both aqueous and organic extracts of V. iphionoides have no cytotoxic effects at the concentrations tested in our experimental conditions.

Discussion

V. iphionoides is a promising medicinal plant from Jordan with a potential anti-inflammatory properties; ²³ however, the effect of V. iphionoides on inflammatory pathways remains poorly understood. To the best of our knowledge, this is the first report showing the anti-inflammatory effects of V. iphionoides in cancerous cells of human origin. We first investigated the effect of this medicinal plant in MRC-5 because they are commonly used to study inflammatory responses. ²³ It has been shown that fibroblasts can produce different types of inflammatory cytokines, which assist in the activation and migration of resident immune cells such as macrophages. ²³ Inflammatory cytokines such as IL-6 stimulate the differentiation of fibroblasts to myofibroblasts, which in turn can produce extracellular matrix proteins leading to fibrosis. Extracellular matrix proteins have the ability to recruit other inflammatory cytokines, and along with the ability of fibroblasts to produce inflammatory cytokines and the activation and migration of macrophages all can contribute to chronic inflammation.^23,24 It is well-established that the bacterial endotoxin LPS is able to induce the production of the pro-inflammatory IL-6 cytokine, and for this reason, LPS is often used in in vitro models of inflammation. ²⁰ LPS induces IL-6 production via the activation of the nuclear factor-kappa B (NFκB); a transcriptional factor that regulates the activity of many pro-inflammatory genes.^20,25 As result of this activation, the levels of pro-inflammatory biomarkers significantly increase in circulation. ²⁶ It is now well known that sustained levels of pro-inflammatory mediators are associated with several pathological conditions including different types of cancer. ²⁷ In the current study, V. iphionoides seems to be able to protect MRC-5 against a dramatic increase of IL-6 levels due to their exposure to the bacterial endotoxin LPS (500 ng/mL) (Figure 1). A similar response was observed by treating the cells with water and methanolic plant extracts at the high concentration of 125 µg/mL; however, the aqueous water seems more potent in reducing LPS-induced IL-6 secretion (Figure 1(a)) compared to the methanolic extract (Figure 1(b)). The different potency exerted by V. iphionoides in MRC-5 could be explained by a different phytochemical composition of the aqueous and organic extract. Our results are in accordance with the study carried out by Budovsky and colleagues (2014). The authors have provided experimental evidence of the anti-inflammatory effect of an organic extract of V. iphionoides that at the high concentration of 100 µM was able to reduce the basal secretion of IL-6 compared to untreated MRC-5. ²⁸ However, in our study, we did not measure the levels of IL-6 in response to V. iphionoides without LPS because we decided to investigate whether this medicinal plant could exert a protective effect against a pro-inflammatory stimulus (LPS).

Inflammation has been associated with prostate cancer, ¹⁵ and the pro-inflammatory cytokine IL-6 seems to play an important role on the progression of this disease.^15,22,24 There are several in vitro studies showing the activity of medicinal plants in reducing IL-6 production, ¹⁵ and how this effect can be also found in prostate cancer cells. For example, andrographolide, which is isolated from a traditional Chinese and Indian medicinal plant, was found to inhibit IL-6 at both gene and protein levels in prostate cancer cells. ²¹ However, there are no previous reports on the in vitro anti-inflammatory effect of V. iphionoides against prostate cancer. Therefore, we tested the hypothesis that V. iphionoides water and methanolic extracts reduced IL-6 secretion in PC3 cells. We found that in PC3 cells, with both water and methanolic extracts, have similar trend of reducing LPS-induced IL-6 release (Figure 2); however, only methanolic extract at 125 µM reached significant levels of reduced LPS-induced IL-6 secretion (Figure 2(b)).

It is important to highlight that the activity of V. iphionoides in reducing IL-6 production in both MRC-5 and PC3 cells was not due its cytotoxicity. Data obtained by performing a viability assay have clearly indicated that the plant extract did not reduce cell growth at any concentration tested (Figure 3). We can hypothesise that the protective effect of V. iphionoides against the significant increase of IL-6 levels induced by the bacterial endotoxin LPS could be related to the modulation of inflammatory pathways. Therefore, the mechanism underlying V. iphionoides-reduced IL-6 production in both MRC-5 and PC3 cells require further investigation. It is possible that V. iphionoides could influence transcription factors, such as NFkB, which are known to regulate IL-6 secretion. Further studies are needed to elucidate the mechanisms underlying the effects of V. iphionoides on IL-6 observed in the current study, and to investigate the downstream events.

Conclusion

This study provides the first in vitro evidence of the anti-inflammatory effect of water and methanolic extract of V. iphionoides in MRC-5 and PC3 cells. Taking into account the key role of inflammation, and mainly IL-6 production, in cancer progression, our results suggest a potential beneficial effect of V. iphionoides in modulating pro-inflammatory responses in human prostate cancer PC3 cells. However, how this observation is relevant in vivo still requires further investigation.

Finally, this study indicates that higher concentrations of V. iphionoides is more effective in preventing the production of inflammatory mediators in both MRC-5 and PC3 cells. However, non-cancerous and cancerous cells seem to respond differently to the plant extract in terms of the extent of the anti-inflammatory effect; water extract was more potent in MRC-5 compared to the methanolic extract which induced a stronger effect in PC3 cells. The phytochemical composition of the aqueous and organic extracts needs to be characterised in order to further understand the different effects observed in human cells.