Evaluation of anticancer activity of Clerodendrum viscosum leaves against breast carcinoma.

INTRODUCTION: The use of natural resources as medicines for cancer therapies has been described throughout history in the form of traditional medicines. However, many resources are still unidentified for their potent biological activities. Clerodendrum viscosum is a hill glory bower reported as a remedy against oxidative stress, skin diseases, and intestinal infections.
MATERIALS AND METHODS: We have collected the C. viscosum leaves and used for the preparation of 70% methanolic extract (CVLME). Then, CVLME has been confirmed for anticancer properties on various cancer cell lines by evaluating cytotoxicity, cell cycle analysis, induction of ROS and apoptosis, and nuclear fragmentation. Further, the phytochemical analysis of CVLME was evaluated through high-performance liquid chromatography.
RESULTS: Cell proliferation assay revealed the selective cytotoxicity of CVLME against breast cancer cell line (MCF-7). The FACS-based cell cycle analysis showed increased subG1 (apoptosis) population dose dependently. Further, the apoptosis-inducing effect of CVLME was confirmed by annexin staining. Flow cytometry and confocal microscopy revealed the selective ROS generation upon CVLME treatment. The confocal-based morphological study also revealed condensed and fragmented nuclear structure in CVLME-treated MCF-7 cells. Phytochemical investigations further indicated the presence of tannic acid, catechin, rutin, and reserpine which might be the reason for the anticancer activity of CVLME.
CONCLUSION: The above-combined results revealed the anticancer effect of CVLME, which may be due to the selective induction of ROS in breast carcinoma.

Introduction

The free radicals are mainly categorized into reactive oxygen species (ROS) and reactive nitrogen species (RNS). Both formed through metabolic reactions and modulate numerous cellular processes. Unfortunately, these are highly reactive molecules, and at high concentrations, they cause damage to lipids, proteins, and DNA which ultimately leads to initiate various detrimental diseases including cancer.[123] Cancer is a state of the body with unrestrained multiplication of cells. It can develop almost everywhere in the human body. Among 36 cancer types, breast cancer is on top with 49.9% incidences and the second-highest 12.9% crude mortality rate.[45] The current treatments for cancer include chemotherapy and radiation therapy, but these therapies are very expensive and can leave the patients with enormous side effects. In recent years, natural products have been widely used for cancer therapies to overcome the cost and side effects of chemo and radiation therapies. Natural products are documented as a major source of drugs in several diseases including cancers. Among the natural resources, plants are rich in various phytochemicals and more than 75% of the drugs against various infections have been isolated from plant resources. The natural antioxidants from plants could alter the microenvironment and modify the behavior of cancer cells[67] and thus may be useful in the treatment of cancer.[8] Major anticancer drugs isolated from plants are irinotecan, paclitaxel, vincristine, and etoposide.[9] Despite the discovery of many chemically prepared as well as naturally originated drugs, it is necessary to discover new anticancer agents with less toxic and more effective in treatment. The genus Clerodendrum is widely distributed and has been reported for its enormous activities.[101112131415]Clerodendrum viscosum (C. viscosum), sometimes called a Clerodendrum infortunatum, is a hill glory bower and enlisted as a prominent medicinal plant in Ayurveda for ages. C. viscosum has been reported as a remedy against various skin diseases, snake bite, scorpion sting, intestinal infections, and kidney dysfunctions.[1617] In this study, we have prepared the 70% methanolic extract (CVLME). Further, CVLME was used to test its detailed anticancer activity against MCF-7.

Materials and Methods

Reagents and chemicals

The HiMedia Laboratories (India) supplied cell culture media (DMEM and F-12), Amphotericin-B, and other Antibiotics. HyClone Laboratories, Inc., UT and Alexis biochemical, San Diego, CA, USA supplied FBS and DCFH-DA, respectively. WST-1 reagent and apoptosis staining kit (Annexin-V FLUOS) were bought from Roche Diagnostics (Mannheim, Germany). Other reagents including DAPI stain and RNAase A were provided by MP Biomedicals (France).

Collection of plant material

Leaves of C. viscosum were collected from the North Bengal University campus and other area near Siliguri, India and validated by Prof. A. P. Das (Taxonomist of the University of North Bengal). The sample was stored with the accession number of 9617 at the Herbarium of the University. The C. viscosum leaves were further processed, and 70% methanol extract was prepared according to a previously published method and named as CVLME.[18]

Cell lines and culture

The WI-38, MCF-7, U87, HeLa, and A549 cell lines were attained from NCCS, India. Cell lines were supplemented with appropriate media. Normal fibroblast (WI-38), human breast adenocarcinoma (MCF-7), cervical carcinoma (HeLa), and glioblastoma (U87) were grown in DMEM. Moreover, F-12 Ham's medium was used to grow lung carcinoma (A549) cell line. All the media were supplemented with appropriate antibiotics and 10% (v/v) FBS. All the cell lines were maintained in a CO2incubator having constant supply of 5% CO2and 37°C temperature.

Cell toxicity with WST-1 reagent

The cytotoxicity of CVLME on various cell lines was assessed according to earlier described protocol[19] using the WST-1 reagent. The reading was measured at 460 nm.

Cell cycle distribution and apoptosis study

The cells (MCF-7) were treated with various doses of CVLME and analyzed the cell cycle and apoptosis inducing effect according to a previously mentioned protocol.[19] The different phases of cell cycle were evaluated on a flow cytometer (FACS Verse). Further, the Annexin Staining kit was used to evaluate the apoptosis-inducing effect of CVLME on MCF-7 cells. The apoptotic population was also analyzed with a flow cytometer (FACS Verse).

Morphological evaluation using DAPI

DAPI stain was used to observe the nuclear morphology upon treatment with CVLME. For that, cells were seeded and incubated with CVLME (200 μg/ml) in a CO2incubator for the period of 48 h. Cells were then incubated with paraformaldehyde (4%) for 20 min for the fixation, followed by cell permeabilization with Triton X-100 (0.5%). Finally, the cells were incubated with 10 μg/ml DAPI solution and observed using a confocal microscope.

Determination of ROS levels

Fluorescent dye DCFH-DA was used to evaluate the effect of CVLME on ROS induction in MCF-7 and WI-38 cells. The cells were incubated with CVLME (0–300 μg/ml) for 24 h and further stained with DCFH-DA. After washing, the cell samples were observed using FACS Verse. In addition, to flow cytometric observations, we have reconfirmed the results of CVLME-induced ROS in MCF-7 using a confocal microscopy. Briefly, 1 × 105 cells were seeded and treated with CVLME (200 μg/ml) for 24 h. After incubation, treated cells were again incubated using fluorescent dye DCFH-DA solution for the period of 30 min at RT. After the proper washing, the coverslip containing cell samples was fixed on a glass slide and analyzed using a confocal microscope.

High-performance liquid chromatography analysis of CVLME

High-performance liquid chromatography (HPLC) was performed to analyze the phytocompounds present in CVLME. The previously published protocol was used for sample preparation, HPLC run, and analysis.[19]

Statistical analysis

Cytotoxicity data were shown as the mean ± standard deviation (SD) of six independent readings. The experiments accomplished using the FACS as the mean ± SD of three independent readings. The statistical analysis was performed using KyPlot version 2.0 beta 15 (32 bit) (KyensLab Inc., Tokyo, Japan). The IC50values were calculated by following previously published protocol.[19]

Results

Cell toxicity test

The toxic effect of CVLME on A549, MCF-7, U87, HeLa, and WI-38 was assessed with the help of WST-1 chromogenic reagent. Figure 1a shows that the CVLME selectively kill breast carcinoma cells (MCF-7) and it was nontoxic to other cancer cell lines as well as noncancerous fibroblast cell line WI-38. The highest dose of CVLME showed 40.15% of cell viability in MCF-7 cells. The graphical representation of IC50values of CVLME against various cell lines are shown in Figure 1b.

Figure 1

Cytotoxic potential of CVLME on A549, MCF-7, U87, HeLa, and normal fibroblast cells WI-38. (a) Cell viability was determined with WST-1 cell proliferation reagent. Results were expressed as cell viability (% of control). (b) The graphical representation of IC50 values of CVLME against indicated cell lines. All data are expressed as mean ± standard deviation (n= 6)

Evaluation of cell cycle phases

MCF-7 cells were treated with CVLME to analyze the cell cycle profile through a flow cytometer. After 48 h of treatment of CVLME, the cell cycle analysis was done and the phase distribution profile showed the induction of apoptotic peak (sub G1 phase) from 50 μg/mL treatment. The highest dose of CVLME showed 15.02% population of sub G1 phase with a slight increase of synthetic (S) phase population in the MCF-7 cell line [Figure 2].

Figure 2

Flow cytometric analysis of MCF-7 cells treated with CVLME. Cell cycle phase distribution of Sub-G1, G1, S and G2/M phases of control MCF-7 cells (a) and treated MCF-7 cells with the indicated doses of CVL (b-f) ME for 48 h

Annexin V/PI staining

The dose-dependent CVLME treatment increases apoptotic population of MCF-7 cells, which was confirmed by the apoptosis staining kit Annexin. Figure 3a shows that the 99.81% population was FITC negative, whereas the apoptotic cells gradually increases with increasing dose. As shown in Figure 3f, the highest dose of CVLME phase distribution 39.91% FITC positive population (early apoptosis).

Figure 3

Apoptosis detection of MCF-7 cells by Annexin V/PI staining with different concentrations of CVLME. (a) 0 μg/ml, (b) 50 μg/ml, (c) 100 μg/ml, (d) 150 μg/ml, (e) 200 μg/ml, (f) 300 μg/ml.

Morphological evaluation through DAPI

As shown in Figure 4, normal intact nuclear morphology was observed in untreated MCF-7 cells. However, abbreviated nuclei were detected on treatment with 200 μg/mL of CVLME.

Figure 4

Morphological Assessment of CVLME-treated MCF-7 cells through confocal microscopy. Nuclei were stained with DAPI and observed under a confocal microscope in comparison to the untreated cells. The white arrows indicate ruptured cells with fragmented nuclei

Measurement of ROS levels

Upon CVLME treatment, the DCFH-DA-stained cells (MCF-7 and WI-38) were observed using a flow cytometer. As revealed from Figure 5a, the CVLME treatment does not induce any ROS amount in normal lung fibroblast cell line WI-38. However, the dose-dependent increase in ROS was detected in treated MCF-7 cells. The highest dose of CVLME increases nearly 3.7 fold of ROS in MCF-7 as compared to control cells. The CVLME-mediated induction of ROS was further reconfirmed through a confocal microscopy. As shown in Figure 5b, a negligible amount of ROS was detected in untreated MCF-7 cells, whereas green fluorescence generated in CVLME (200 μg/mL) treated MCF-7 cells indicated the ROS generation.

Figure 5

ROS analysis of CVLME treated MCF-7 and WI-38 cells through FACS and confocal microscopy. (a) Intracellular ROS levels were examined under FACS using the DCFH-DA staining and presented graphically for CVLME treatment in MCF-7 and WI-38 cells. (b) Intracellular ROS was visualized under the confocal microscope with ×400 magnification for CVLME treatment against MCF-7 cells

High-performance liquid chromatography analysis

The HPLC analysis was performed to identify the probable bioactive compounds present in CVLME. As shown in Figure 6, the HPLC evidence the presence of various phytochemicals including tannic acid, catechin, reserpine, and rutin.

Figure 6

High-performance liquid chromatography chromatogram of CVLME. (a) Peaks marked signify the retention peak of CVLME matched with the retention time of the known bioactive compounds in the same condition. (b) Chemical structure of the compounds identified through high-performance liquid chromatography

Discussion

In the past two decades, the scientific community has investigated treatment against many diseases using improved scientific technologies. However, some diseases including cancer remain elusive, especially from a therapeutic view. Natural resources have been playing a very significant part in the finding of new anticancer drugs. Among the natural resources, medicinal plants are the major contributors to drug development. To date, medicinal plants are the largest natural resource for the improvement of active anticancer candidates.[202122]C. viscosum is a widely distributed medicinal plant and has been stated previously for the various potent activities. In this, we used the leaves of plant and prepared the 70% methanolic extract and tested its anticancer effect using various cancer cell lines. WST-1 is reduced to a chromogenic product formazan by cellular mitochondrial dehydrogenases generally produced by the viable cells.[23] The selective targeting of cancer cells is the key principle behind the development of anticancer drugs. CVLME also showed the selective targeting of MCF-7 cells. An ideal anticancer drug should inhibit the growth of cancer cells by arresting the cells in various cell cycle phases, and it has been evaluated. In recent years, the flow cytometer is widely used to determine the DNA content of the cells and the number of cells following the process of apoptotic.[24] From the results, it was deciphered that the CVLME selectively increases sub G1 population (apoptosis) with a slight increase in the synthetic (S) phase. The process of necrosis is usually occurred by disturbing the cellular architecture-like breakage of plasma membrane (PM), and in contrast, the process of apoptosis occurs without damaging the integrity of PM.[25] The PM is a bilayer structure and mostly composed of lipids and proteins. Phosphatidylserine (PS) is a phospholipid and presents mainly at the inner leaflet of PM. The two enzymes, namely, flippases and floppases regulate the translocation of these phospholipids through the membrane.[26] When a cell decides to undergo the process of apoptosis, it initiates the translocation of PS to the outer membrane. In the present study, CVLME-induced cell death was observed with the Annexin-V-FLUOS Staining kit to distinguish between apoptotic and necrotic populations. The assay is based on the observation of PS, which is located at the cytoplasmic side of PM and translocate to the surface of the cell during the initiation of apoptosis and this translocation of PS can be observed using the Annexin-V staining dye.[27] FITC-labeled Annexin-V (+) cells are detected by flow cytometry in combination with propidium iodide allowing characterization of the progressive stages of apoptosis. The results from Figure 3 showed the early apoptosis started in MCF-7 from 50 μg/ml dose of CVLME. The highest dose of CVLME showed 39.91% of apoptotic population. During the process of apoptosis, enzymes such as endonucleases such as caspase 3-activated DNase (CAD) play an crucial role in initiation of DNA cleavage.[28] The characteristic changes, particularly in the nucleus were observed through a nuclear staining with the fluorescent dye DAPI.[29] As shown in Figure 4, the intact nuclear morphology was witnessed in untreated MCF-7 cells. However, the disturbed nuclear morphology with fragmentation was observed in 200 μg/ml CVLME treated MCF-7 cells. The cellular ROS levels also impaired the proliferation of cells.[30] To evaluate the effect of CVLME treatment of MCF-7 cells, we used the 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. It penetrates into the cells and converted to its fluorescent form and which was further analyzed by the flow cytometer and confocal microscopy.[3132] The result showed the negligible ROS levels in CVLME treated normal WI-38 cells and the levels were very much elevated in MCF-7 cells upon CVLME treatment. A similar result was observed upon visualization under a confocal microscope. A review published by Wang et al. reported the list of almost 300 phytocompounds isolated from Clerodendrum genus plants.[17] Among the Clerodendrum genus, C. viscosum is one of the most studied medicinal plants for various biological activities. The presence of clerodone, viscosene, scutellarin, and hispidulin-7-0-glucuronide has been previously reported in Nandi et al. Moreover, Shendge et al. have previously isolated the ellagic acid[33] and apigenin[34] from the leaves of C. viscosum. In the present study, we have performed HPLC analysis and Figure 6 shows the presence of various phytocompounds such as catechin, tannic acid, reserpine, and rutin in the CVLME extract. Catechin was previously established for potent antioxidant activities.[35] Tannic acid was shown previously for its activity against numerous degenerative diseases.[36] Furthermore, reserpine and rutin were shown for antioxidant and anticancer properties.[37] Recently, the ROS-elevated therapy is gaining attention in cancer treatments. Many drugs have been reported previously for their anticancer activity by inducing ROS in cancer cells.[34383940] Moreover, many reports have supported the ROS inducing potential of catechins[41] and tannic acid.[4243] In the present study, using flow cytometry and confocal microscopy-based experiments, we have observed that CVLME induces ROS in MCF-7 cells dose dependently [Figure 5]. The ROS-inducing mechanism of CVLME may be due to the presence of catechin, tannic acid, and other phytochemicals present in it. The presence of such phytochemicals in CVLME possibly increases the genomic instability and further induces apoptosis in MCF-7 cells, as confirmed by the annexin V/PI staining [Figure 3] and confocal microscopy [Figure 4].

Conclusion

The present finding demonstrates the anticancer potential of CVLME against MCF-7 cells. The cell proliferation assay reveals the toxic nature of CVLME against MCF-7 cell line but not toward other cancer cell lines and WI-38. Moreover, the flow cytometric data demonstrated the treatment of CVLME increased sub G1 population and induced early apoptosis in MCF-7 cells. Microscopic observations revealed the nuclear fragmentation and selective induction of ROS upon CVLME treatment. The selective ROS induction along with the presence of bioactive phytochemicals may be responsible for the cytotoxic nature of CVLME against MCF-7 cell line. In future, additional investigation requires to establish the exact anticancer mechanism of CVLME against breast cancer.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.