In Vitro Anticancer Activity of Syzygium calophyllifolium on A549 Lung Cancer Cells

Rahul Chandran and Heidi Abrahamse

26.1 INTRODUCTION

Cancer has become one of the most studied type of diseases, and the reason for this being the high mortality and incidence reported every year. The condition is characterized by the irregular proliferation of cells that leads to life threatening events (Roy A et al. 2017). According to recent cancer statistics, 18.1 million cancer cases were reported with an estimated mortality of 9.6 million in 2018 (Bray et al. 2018). The statistics also reported a high incidence of lung cancer in both sexes (11.6%), which led to the highest number of cancer death cases (18.4%) as compared to other cancer types. Small cell and non-small cell are the histologic classifications of lung cancer, with both having common symptoms like cough, systemic problems, hemoptysis, dyspnea, etc. A diagnosis at the early stages can improve the lifestyle of the patient for a prolonged period (Latimer and Mott 2015). With several reported factors causing cancer including genetic and lifestyle changes, alternative treatment methods with high efficiency are sought after at an alarming demand. Science has been trying all the possible treatment modalities to eradicate cancer since its outburst. However, due to certain limitations and side effects of currently available treatments, science has been developing the idea of treating cancer using plant-based medicine minimizing the side effects and increasing the therapeutic efficiency.

Plants are of common interest among traditional healers since ancient times and are being studied by researchers across the world for their medicinal properties. Plants are a well chosen alternative mode of treatment against cancer in many countries, with around 3000 plants already reported to have an effective potential to fight against cancer (Alves-Silva et al. 2017; Tariq et al. 2017). Natural products, especially those from plants, have gained interest due to their biologically active compounds and their chemotherapeutic effects against cancer. They are biologically friendly and less or non-toxic to normal cells (Mishra and Tiwari 2011). The ability to induce alternative modes of cell death makes these naturally derived anti-cancer drugs a promising candidate for anti-cancer research. Moreover, there is evidence that natural product- derived anti-cancer drugs can induce alternative modes of cell death pathways (Khalid et al. 2016; Gali-Muhtasib et al. 2015).

The genus Syzygium is being well studied for its medicinal properties by natural scientists. One of the authors (Chandran) has also performed several biochemical and pharmacological studies on species of Syzygium (Chandran et al. 2015, 2016,

  • 2017a, 2017b, 2017c, 2018; Sathyanarayana et al. 2018). All of these studies have proven the tremendous therapeutic potential of Syzygium calophyllifolium Walp. The plant has also been proven to be a good remedy for toothaches and inflammation (Rajan et al. 2005; Sathyavathi and Janardhanan 2011). The present study examines the effect of the Syzygium calophyllifolium bark methanol (SCBM) extract on A549 lung cancer cells to facilitate an alternative treatment modality.
  • 26.2 MATERIALS AND METHODS
  • 26.2.1 Collection of Plant Materials

The bark was collected during the month of February 2012 from Ooty, India. The collected plant material was identified, and its authenticity was confirmed by comparing the voucher specimen at the herbarium of the Botanical Survey of India, Southern Circle Coimbatore, Tamil Nadu (BSI/SRC/5/23/2012- 13/Tech.453). The freshly collected plant material was cleaned to remove adhering dust and then dried under shade. The dried sample was powdered and used for further studies.

26.2.2 Successive Solvent Extraction

The air-dried, powdered tree bark (100 g) was extracted in a Soxhlet extractor using methanol (300 mL), vacuum dried in a rotary vacuum evaporator, and used for further studies.

26.2.3 Cell Culture and Treatment

A commercially purchased A549 lung cancer cell line (ATCC CCL-185) was used for the anti-cancer study. Approximately 5 x 10s cells were seeded in 3.4 cm2 diameter culture dishes and cultured in a Roswell Park Memorial Institute 1640 Medium (Sigma-Aldrich, R8758), supplemented with 10% fetal bovine serum (Gibco, 306.00301) and 1% antibacterial (penicillin) agents. The culture was maintained at 37°C with 5% carbon dioxide (C02) and 85% humidity for 4 hours to allow the cells to attach. Culture dishes with more than 90% confluence were used for these experiments. The experimental groups were divided into untreated controls and the cells treated with SCBM at three different doses (5, 10, and 20 pg/mL).

26.2.4 Cellular Morphology-Inverted Microscopy

The morphology of the SCBM extract (5, 10, and 20 pg/mL)- treated cells was analyzed after 24 hours of incubation using an inverted light microscope (Wirsam, Olympus CKX41). Once the digital images were recorded, the cells were tryp- sinized using 1 mL/25cm2 of TrypLE Express (Invitrogen, 12605-028) and resuspended in Hank’s Balanced Salt Solution to perform further assays.

26.2.5 Cellular Proliferation-Adenosine Triphosphate Luminescent Assay

The CellTiter-Glol luminescent assay (Promega, G7571, Anatech Analytical Technology, Bellville, South Africa) is a homogeneous method for determination of cellular proliferation and quantification of the adenosine triphosphate (ATP) present in the metabolically active cells. An equal volume (50 pL) of the reconstituted ATP reagent and the cell suspension was mixed on a shaker for 2 minutes to induce cell lysis, followed by incubation at room temperature for 10 minutes in the dark to stabilize the luminescent signal. The luminescent signal was read using the 1420 Multilabel Counter Victor3 (PerkinElmer, Separation Scientific).

26.2.6 Cytotoxicity-Lactate Dehydrogenase Assay

The membrane integrity was assessed by estimating the amount of lactate dehydrogenase (LDH) present in the culture media. The cytosolic enzyme LDH will be released into the media due to membrane damage. The CytoTox 96® X assay (Anatech, Promega G 400) was used to measure the LDH released. An equal volume (50 pL) of reconstituted LDH reagent and cell culture medium was mixed and incubated in the dark at room temperature for 30 minutes. The colorimetric compound was measured spectrophotometrically at 490 nm (PerkinElmer, Victor3™)-

26.2.7 Hoechst Stain

The damage caused by the SCBM extract was analyzed using Hoechst nuclear stain. The MCF-7 cells were cultured in

  • 3.4 cm diameter culture dishes over sterile cover slips and allowed to reach a confluence of 80%. The cells were then treated with different concentrations of SCBM (5, 10, and 20 pg). After 24 hours of incubation, the cells were stained with 1 pg/mL Hoechst stain (Hoechst 33258. H21491) for 30 minutes. Thereafter, the cells were rinsed with Phosphate Buffer Saline (PBS), and the blue fluorescent signal was examined using the Olympus fluorescent microscope.
  • 26.2.8 Statistical Analysis

All the results were expressed as mean ± SEM. The statistical significance was determined by using the one-way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison tests, p < 0.05 was considered statistically significant.

26.3 RESULTS AND DISCUSSION

This research was an attempt to explore the possibility of using drugs of natural origin against lung cancer. The methanol extract of SCBM was treated and analyzed on A549 cells in vitro. The cytotoxic effects of potentially active drugs can be determined using various qualitative and quantitative biochemical assays. In the present study, the cell morphology, ATP, LDH, and nuclear damage using Hoechst stain were studied.

26.3.1 Cellular Morphology

The change in cellular morphology was observed using inverted microscopy. The A549 cells were treated and incubated with different doses of the SCBM extracts. An evident change in cellular morphology was observed after 24 hours. The loss of membrane integrity, cell to cell contact, rounding, vacuolation, and cell detachment for the culture plate surface were seen as indications of cell death. These signs were more evident in the treated groups as compared to the control (untreated) ones (Figure 26.1). These morphological features depicted the toxicity of the SCBM extract on A549 cells, indicating the signs of apoptosis (Monga et al. 2013). However, this has to be confirmed using further biochemical and molecular study.

26.3.2 ATP Cell Metabolism Assay

Mitochondria generate ATP as an energy molecule for their metabolic activities, and any damage to this organelle will result in the imbalanced ATP production and ATP-dependent mechanisms necessary for cell functions (Maurer and Meyer 2016). ATP is one of the indications of healthy cell metabolism, which can effectively be assessed using a luminescent quantification assay. As observed in the cell morphology, Figure 26.2 expresses higher cellular metabolism in the control group, with the least ATP in 20 |ig/mL of the SCBM extract.

Morphology of A549 cells after the SCBM extract treatment, (a) Control

FIGURE 26.1 Morphology of A549 cells after the SCBM extract treatment, (a) Control: (b) SCBM 5 pg; (c) SCBM 10 pg; and (d) SCBM 20 pg (lOx objective).

ATP metabolism in A549 cells treated with the SCBM extract. The data represent the mean ± SEM. Significantly different

FIGURE 26.2 ATP metabolism in A549 cells treated with the SCBM extract. The data represent the mean ± SEM. Significantly different

at ***p < 0.001 when compared to the control S. calophyllifolium bark methanol (SCBM) extract.

A significant reduction in the cell proliferation was observed in a dose-dependent manner (5, 10, and 20 pg/mL) as compared to the control. As the concentration of the extract was increased, the ATP levels were also decreased. The results of the present study support the findings previously reported by one of the authors (Chandran) in breast cancer (MCF-7) cells (Chandran et al. 2018).

26.3.3 Cytotoxicity-LDH Assay

LDH is a cytoplasmic enzyme which is released into the extracellular space upon plasma membrane damage and is found in nearly all types of cells (Gurunathan et al. 2015). The results of the LDH assay are in agreement with the ATP and morphology studies. The loss of cell membrane integrity released the LDH to the culture media when treated with the SCBM extract. The toxic effect of the extract was found best in the highest dose of the extract (Figure 26.3). The comparable levels of LDH implicate the cytotoxic effects of the extract than the untreated control as shown in Figure 26.3. The results of the LDH in the media were directly proportional to the dose of the extract. Xia et al. (2007) reported a correlation between the LDH upregulation and the induction of apoptosis.

26.3.4 Hoechst Stain

Hoechst stain helps to identify the extent of nuclear damage induced by the SCBM extract. The cells responded variably to the doses of the extract, wherein the highest dose showed visible nuclear damage. The nucleus of the control cells stained uniformly, signifying its intact and dense nature. The nucleus of the extract-treated cells showed nuclear margination, chromatin condensation, and deformation of the nuclear size and integrity as compared to the untreated control (Figure 26.4).

LDH levels in A549 cells treated with the SCBM extract

FIGURE 26.3 LDH levels in A549 cells treated with the SCBM extract. The data represent the mean ± SEM. Significantly different at ***p < 0.001 when compared to the control S. calophyllifolium bark methanol (SCBM) extract.

Nuclear damage study in A549 cells using Hoechst stain, (a) Control

FIGURE 26.4 Nuclear damage study in A549 cells using Hoechst stain, (a) Control: (b) SCBM 5 |ag; (c) SCBM 10 |ag; and (d) SCBM 20 |ig (40x objective).

The fluorescent images of the nucleus also supported the fact that the extract promoted DNA damage and cell death.

26.4 CONCLUSION

Natural drugs have gained interest among a majority of the population around the globe. To complement this, the present study was designed to formulate the cytotoxic activity of the SCBM extract against lung cancer cells in vitro. The morphology of the treated cells explained the degree of toxicity induced by the SCBM extract. The reduced ATP and increased LDH levels also implicated the cytotoxic effect of the extract toward A549 cells. The proposed activity of the extract in this study is never a replacement for drugs popularly used for the treatment for lung cancer in-fact a recommendation on how to utilize such natural products for the treatment with best check on diseases like cancer and lesser side effects.

ACKNOWLEDGMENTS

This work is based on the research supported by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (Grant No 98337), as well as grants received from the University of Johannesburg (URC), the National Research Foundation (NRF), and the Council for Scientific and Industrial Research (CSIR)-National Laser Centre (NLC) Laser Rental Pool Programme.

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