In vitro Anticancer Activities of Ethanolic Extracts of Dendrobium crepidatum and Dendrobium chrysanthum against T-cell lymphoma

Background: The orchid plants of Dendrobium, belong to the largest family of flowering plants orchidaceae. The genus Dendrobium reported to possess various therapeutic activities including anticancer activity. In the present scenario, cancer is one of the most leading causes of death worldwide. Therefore, the main objective of the present study was to demonstrate the cytotoxic and apoptotic inducing activity of Dendrobium crepidatum and Dendrobium chrysanthum ethanolic extracts in T-cell lymphoma.


Introduction
Orchid belongs to the family orchidaceae, one of the largest families of flowering plants with nearly 20,000 species and comprising of more than 850 genera [1]. Dendrobium is a huge genus of orchids and contains about 1,200 species. Most of the Dendrobium species grow in the high and mountainous areas, at mild temperature, and in a humid environment. They are characterized by a broad geographical distribution, with different morphological features and are widely distributed in Asia, Australia and Europe, mainly, in India; Sri Lanka; China; Japan; Korea; New Guinea and New Caledonia [2]. Dendrobium genus was found to possess useful therapeutic activities like anticancer, hypoglycaemic, antimicrobial, immunomodulatory, hepatoprotective, antioxidant, and neuroprotective activities. Different array of chemical compounds were reported in orchids including alkaloids, bibenzyl derivatives, flavonoids, phenanthrenes and terpenoids [1]. Cancer is the main cause of death worldwide. Cancers may be caused due to incorrect lifestyle, genetic predisposition and environment. Environmental carcinogens; including both natural and manmade chemicals, radiation, and viruses are the major cause of human cancers now a days. Discovery of medicinal plant drug continues to provide an important leads against various pharmacological targets including cancer, HIV, Alzheimer's, malaria, and pain. It has been confirmed by World Health Organisation (WHO) that herbal medicines serve the health needs of about 80 percent of the world's population; especially for millions of people in the vast rural areas of developing countries [3]. There are various reports of antitumour activity of Dendrobium genus till date. The antitumor effects of inhibitory mechanisms in SNU-484 cells and induced apoptosis through downregulation of Bcl-2 and upregulation of Bax in cancer cells [4,5]. The bibenzyl isolated from Dendrobium draconis inhibited several cancer cell lines including non-small-cell lung cancer cells [6]. There are also reports of antitumor and antibacterial activities from the extracts of Dendrobium nobile [7]. Moscatilin, a bibenzyl derivative from Dendrobium pulchellum found to inhibit the lung cancer cell motility and invasion through suppression of endogenous reactive oxygen species [8]. We have also earlier reported about the antitumour activity of ethanolic extract of an Indian orchid, Dendrobium formosum against T-cell lymphoma [9]. The medicinal properties reported involving different Dendrobium species intrigue us to examine two other species of Indian orchids, regarding their antitumour activity. The two species are Dendrobium crepidatum and Dendrobium chrysanthum whose antitumour activity has not been reported yet to the best of our knowledge. Polysaccharides isolated from stems of Dendrobium nobile, showed a strong antitumor action in inhibiting sarcoma 180 in vivo and HL-60 cells in vitro [4]. In addition, denbinobin isolated from stems of Dendrobium nobile exhibited antitumor action by inhibition of SNU-484 cells and induced apoptosis through downregulation of Bcl-2 and upregulation of Bax in cancer cells [5]. The bibenzyl isolated from Dendrobium draconis inhibited several cancer cell lines including non-small-cell lung cancer cells [6]. There are also reports of antitumor and antibacterial activities from the extracts of Dendrobium nobile [7]. Moscatilin, a bibenzyl derivative from Dendrobium pulchellum found to inhibit the lung cancer cell motility and invasion through suppression of endogenous reactive oxygen species [8]. We have also earlier reported about the antitumour activity of ethanolic extract of an Indian orchid, Dendrobium formosum against T-cell lymphoma [9]. The medicinal properties reported involving different Dendrobium species intrigue us to examine two other species of Indian orchids, regarding their antitumour activity. The two species are Dendrobium crepidatum and Dendrobium chrysanthum whose antitumour activity has not been reported yet to the best of our knowledge. Thus, the present study was undertaken to investigate their antitumor property in T-cell lymphoma.

Plant material collection and preparation of ethanolic extract
Dendrobium crepidatum (D. crepidatum) and Dendrobium chrysanthum (D. chrysanthum) plant was identified and collected from Meghalaya; India. The Leaves of the plants were washed with distilled water and were air-dried. The dried leaves were grinded into fine powder and suspended in absolute ethanol at room temperature for up to7 days, followed by filtration. Then the filtrate was reduced into the form of residue. Finally this extract was collected and stored at 4°C. The residue (extract) was dissolved in sterile distilled water immediately before use as discussed previously [9].

Dalton's lymphoma
Dalton's lymphoma (DL), a murine transplantable T-cell lymphoma was used as a tumour model to study the anticancer activity of the ethanolic extracts. It was originated at the National Cancer Institute, Bethesda, USA, in 1947 in the thymus gland of a DBA/2 mouse and from then it is being serially transplanted in the intraperitoneal cavity from mouse to mouse [10]. For in vitro experiments, 1 × 10 6 DL cells were transplanted intraperitoneally into Swiss albino mice and Dalton's lymphoma ascites (DLA) were maintained from mouse to mouse. DL cells harvested were cultured in RPMI-1640 medium supplemented with 10% FBS and antibiotic solution (penicillin 1000 IU and streptomycin 10 mg/mL) in 5% CO 2 at 37°C. The protocols of the animal study were approved by the central animal ethical committee (CAEC) of the university and the ethic number (Dean/12-13/CAEC/210).

In vitro cytotoxicity assay
The MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide] assay is a quantitative assay, to check viability, toxicity, proliferation and cellular activity of the cells. 3 × 10 4 DL cells were harvested from DL bearing mice and seeded in culture plates in complete RPMI-1640. The cells were treated with various concentration of D. crepidatum and D. chrysanthum ethanolic extract for 24 h at 37°C and 5% CO 2 . On completion of incubation, MTT (5 mg/mL) 10 µL was added into each well followed by incubation for 2 h at 37°C. The purple formazan crystal formed was then dissolved in 100 µL of DMSO at 37°C and left for 30 min. The absorbance was measured in a plate reader at the wavelength of 570 nm [11]. All experiments were carried out in three replicates and the inhibitory concentration (IC 50 value) of the extract was estimated as described earlier [9].

Measurement of reactive oxygen species (ROS)
The intracellular ROS level was analyzed using 2',7'-dichlorofluorescin diacetate (DCFH-DA), an oxidation-sensitive fluorescent dye. Thus to verify the generation of ROS by the ethanolic extracts this assay was performed. 0.5 × 10 6 DL cells were suspended in complete medium and treatment with 250 µg/mL and 325 µg/mL of D. crepidatum and 350 µg/mL, 400 µg/mL of D. chrysanthum ethanolic extract was done for 16 h and 24 h at 37°C. After the incubation, 10 µM of DCFH-DA was added to the cells and incubated further for 30 min at 37°C. The cells were then mounted in PBS on a clean slide, and were observed under fluorescence microscope. The amount of DCF fluorescence intensity exhibited reveals the amount of ROS generation in the cells. The fluorescence induced by DCF was observed [12] and captured using a fluorescence microscope (Nikon E800, Japan).

Apoptotic detection by acridine orange and ethidium bromide staining
Acridine orange/ethidium bromide (AO/EB) dual staining is used to study the, apoptotic and necrotic cells to visualize the cellular and nuclear changes with respect to live cells morphology. For this assay, 0.5 × 10 6 DL cells were treated with indicated concentrations of D. crepidatum and D. chrysanthum ethanolic extracts for 16 h at 37°C. The cells were initially washed with PBS and then stained with ethidium bromide (100 µg/ml) and acridine orange (100 µg/ml) as described earlier with some modification [9]. Post staining the cells were washed with PBS and mounted on a clean glass slide. The cells were examined under a fluorescence microscope (Nikon E800, Japan) and photographed.

Apoptotic nuclear morphology analysis by Hoechst 33342 and propidium iodide staining
To detect the altered nuclear morphology during apoptosis Hoechst 33342/Propidium iodide staining was done. The blue fluorescent Hoechst 33342 is a cell permeable nucleic acid binding dye where as the red-fluorescent propidium iodide (PI) is a cell impairment DNAbinding dye, which enters the cells with lost membrane integrity. 0.5 × 10 6 DL cells harvested were suspended in complete medium, and the treatment was done with D. crepidatum and D. chrysanthum ethanolic extracts at indicated concentration for 16 h at 37°C. The treatment was followed by washing the cells with PBS and staining with Hoechst 33342 (10 µg/ml) and Propidium iodide (15 µg/ml) for 30 min at 37°C [13]. Cells were then studied under a fluorescence microscope and the images got were captured (Evos FL, life technologies).

DNA Fragmentation Assay
DNA fragmentation assay was done to ascertain nuclear fragmentation during apoptosis. Briefly, 1 × 10 6 DL cells were exposed with indicated concentrations of D. crepidatum and D. chrysanthum ethanolic extracts for 16 h at 37°C. The cells were washed followed by cell lysis using lysis buffer (20 mM TrisHCl, pH 8.0, 5 mM EDTA, 40 mM NaCl, and 1% SDS) at 0°C for 15-30 min. The lysate obtained was spinned at 3000 rpm for 8 min. The supernatant was transferred in a tube followed by incubation with Proteinase K (20 mg/ml) for 2 h at 37°C. The DNA obtained was precipitated with absolute ethanol and 5 M NaCl at -20°C overnight. To pellet out the DNA, centrifugation was done and the pellet was washed with 70% ethanol. The DNA was air dried and dissolved in distilled water, as discussed earlier [9]. Now to check the DNA fragmentation, agarose gel (1.8%) was run and the DNA band pattern was observed in a gel doc (G: Box, Syngene).

Cell cycle analysis
Flow cytometry was done to analyze the cell cycle distribution of DL cells on exposure to both the ethanolic extracts. Briefly, DL cells (1×10 6 / mL/6 well plate) were treated with the ethanolic extracts at indicated concentrations for 24 h. On completion of incubation, cells were harvested after washing with PBS. Now the cells were fixed with 70% ethanol overnight at -20°C followed by washing with cold PBS. The cells were then subjected to staining with 500 μL PI-RNase solution (1 mg/mL PI solution, Triton X-100 (0.1% v/v), and 10 mg/mL RNAse) at 37°C for 30 min in dark [14]. Finally, the cells were analyzed for DNA contents on a flow cytometer FACScan (Becton Dickinson, NJ, USA). The cell cycle histograms were analyzed using Cell Quest software (Becton Dickinson).

Statistical analysis
Data obtained were presented as mean ± standard error mean (SEM) of at least three independent experiments. The statistical analysis of data was performed with one-way analysis of variance (ANOVA) followed by Bonferroni -test and values < 0.05 were considered significant using Sigma Stat 2.0 version.

In vitro cytotoxicity
The cytotoxic activity of the two plant extracts were measured using the MTT assay against Dalton's lymphoma. The cytotoxicity was assessed at 24 h following treatment with different concentrations of the ethanolic extracts in the range of 100-500 μg/mL. The findings showed that, the extracts inhibited the growth of DL cells in a dose dependent manner ( Figure 1A and 1B). The extract of D. crepidatum was most active against DL cells with IC 50 value of 325 μg/mL, while the extract of D. chrysanthum was active against DL cells at IC 50 value of 400 μg/mL. Thus, we can conclude that both the extracts induce cytotoxic effect against DL cells. (Figure 1)

Acridine orange/ethidium bromide staining
Acridine orange/ethidium bromide (AO/EB) staining is performed to observe the morphological changes associated with apoptotic cell death. Acridine orange (AO) is a vital dye that could stain nuclear DNA with an intact cell membrane and ethidium bromide (EB) could only stain cells that had lost membrane integrity [15]. Untreated (control) cells displayed normal green nucleus with intact membrane. The cells treated with indicated concentrations of D. crepidatum and D. chrysanthum for 16 h showed marked changes in morphology such as irregular shape, membrane blebbing, and the early apoptotic cells displayed greenish yellow nucleus with condensed chromatin whereas the late apoptotic cells showed orange red nucleus with chromatin condensation and nuclear fragmentation Our data also showed a dose dependent increase in apoptotic cell death when compared to control in case of both the extracts (

DNA ladder assay
The ladder like DNA fragmentation is qualitative indicator of apoptosis. Therefore, to elucidate further, whether the plant extracts decreased cell survival by the induction of DNA fragmentation during apoptosis, DNA ladder assay was done. In the present study, no ladder like pattern was observed in control cells whereas typical ladder like pattern was observed on treatment with the extracts. On treatment for 16 h treatment it showed typical ladder like pattern as visible with later stage apoptosis (Figure 3). This implied that, incubation of DL cells treated with both the extracts for 16 h induced cell death which was markedly accompanied by the presence of DNA fragments. This observation also validates the results from the study of apoptotic cell death by fluorescence staining which showed nuclear fragmentation in apoptotic cells, dotted red arrow shows membrane blebbing in early apoptotic cells and yellow arrows shows necrotic cells].

Cell cycle analysis
To study whether the ethanolic extract of D. crepidatum and D. chrysanthum could stimulate antitumor activity due to delay or arrest in the cell cycle phases, cell cycle distribution was analysed by FACS in asynchronous DL cells. Flow cytometry analysis exhibited an increase in the percentage of cells in G2/M phase treated with the ethanolic extracts compared to control ( Figure 6A

Discussion
Natural products play an important role in remedial of several ailments. The main objective of the present study was to evaluate the   [16]. Thus, our result advocates that both the ethanolic extracts were capable of inducing cytotoxicity in Dalton's lymphoma. It has been reported that ROS metabolism play an important role in all types of programmed cell death [17]. It has also been reported that low and intermediated levels of ROS can protect cell from apoptotic cell death by activating antioxidant mechanisms, on the contrary, high levels of intracellular ROS results in a breakdown of the transmembrane mitochondrial potential (ΔΨm), commonly occurring during early apoptosis, which is followed by DNA fragmentation regardless the type of cell and the apoptotic signalling [18]. Thus, to investigate the level of ROS in ethanolic extract treated cells; DCFH-DA dye was used, which showed increased ROS generation in DL cells compared to untreated cells (control). It has been reported that HeLa cells exposed to methanolic extract of B. racemosa bark (MEBR) and then exposed with DCFH-DA showed bright green fluorescence due to induced ROS production and its intracellular accumulation [19]. Thus our result suggests that an increase in the intracellular levels of ROS might lead to cellular damage and could induce apoptotic cell death by the ethanolic extracts in DL cells. Whether the ethanolic extracts could induce apoptosis, the morphological character changes in DL cells were investigated using the AO/EB staining and Hoechst 33342/PI staining under fluorescence microscope. We observed that treatment for 16 h showed presence of early as well as late apoptotic cells. Early apoptotic cells showed membrane blebbing with greenish/yellow condensed chromatin and late apoptotic cells showed more condensed and fragmented chromatin (orangish/red); at higher concentrations few necrotic cells were also observed with intact deep orange/red nucleus. We have earlier reported similar observations with D. formosum ethanolic extract treated Dalton's lymphoma with a dose and time dependent apoptotic cell death [9]. Induction of apoptosis is the key to success of plant products as anticancer agents [20,21]. On staining with Hoechst 33342/PI, after 16 h of treatment with D. crepidatum and D. chrysanthum ethanolic extracts, also showed dose dependent apoptotic cell death with presence of characteristic apoptotic nuclei. Cells which are undergoing apoptosis typically exhibit fragmentation of the DNA into small oligonucleosomal fragments, a hallmark of apoptosis [22]. Hence, to further validate the nuclear fragmentation occurring during apoptotic cell death as observed with fluorescent staining, analysis of DNA fragmentation was done. The results obtained confirmed that both the ethanolic extracts induced DNA ladder formation, which is the characteristic feature of apoptosis. In our previous study, similar DNA ladder pattern was observed on treatment of DL cells with D. formosum ethanolic extract [9]. Typical ladder pattern due to induction of apoptosis was also reported with aqueous ethanol seed extract of Ziziphus mauritiana in HL-60 cells in a concentration and timedependent manner [23]. The cell cycle studies were done to rule out whether the ethanolic extracts affects the cell cycle progression or not and our analysis showed that they might be inhibiting the growth of DL cells by arresting them at G2/M phase. In our previous study with D. formosum ethanolic extract, we have reported similar results where the extract is found to arrest the DL cells at G2/M phase [9]. In another report, it was shown that moscatilin from Indian orchid Dendrobium moschatum and Dendrobium loddigesii also induces arrest of HCT-116 cells at G2/M phase of cell cycle [24]. Thus our present data are in corroboration with the above findings and they further substantiate the inhibition of lymphoma cells by antitumor activity of the ethanolic extracts.

Conclusion
The results from our study evidently demonstrated the cytotoxic and apoptosis inducing activity of the ethanolic extract of D. crepidatum and D. chrysanthum against Dalton's lymphoma. Thus, both the ethanolic extracts seem to possess profound cytotoxic activity against cancer cells. As apoptosis as well as delay in cell cycle are regarded as a main target in discovery of anticancer agents, therefore results obtained above confirm the potential of D. crepidatum and D. chrysanthum as a novel chemotherapeutic agent. But the molecular mechanistic aspects of its effect are still unidentified. Thus, further research work is needed to establish the exact anticancer activity of both the ethanolic extracts.