Anticancer property of Colchicine isolated from Indigofera aspalathoids

L. Krishnasamy¹*, M. Masilamani Selvam¹, Bharathi Ravikrishnan²

¹Department of Biotechnology, Sathyabama University, Rajiv Gandhi Salai, Chennai - 600119, India

²Department of Biotechnology, Hindustan College of Arts & Science, Chennai, India

*Corresponding Author E-mail: lksamy2004@gmail.com

ABSTRACT:

The chief goal of our current research was to establish the anticancer property of Colchicine, a fresh compound isolated from Indigofera aspalathoids. Hep3B cell lines were treated with Colchicine for 24 hrs, 48 hrs and 72 hrs. MTT assay was performed to check the degree of cytotoxicity. The morphological changes of colchicine treated cell lines and control groups were observed using phase contrast microscope. Acridine Orange/Ethidium Bromide (AO/EB) double staining, Propidium iodide (PI), were done to check apoptosis of Hep3B cell lines. Cell cycle analysis was employed to determine apoptosis in Hep3B cell lines and degree of apoptosis was quantified using flow cytometry. Colchicine induces cell death in Hep3B cell lines in a dose dependent manner. The IC50 (half maximal inhibitory concentration) value of colchicine on Hep3B cells was calculated as 344.25 µg/ml. Control group cell lines showed normal morphology, whereas the Colchicine treated cells showed altered morphology, also cells were not properly adhered to the culture flask. Colchicine induces apoptosis in Hep3B cell lines. Thus, it could be used as an active member of pharmaceutical or therapeutic concerns for the drug development.

KEYWORDS: Colchicine, Hep3B cell line, Indigofera aspalathoids, Cytotoxicity assay, Cell cycle assay, Apoptosis.

INTRODUCTION:

Cancer, a dreadful disease, has the potentials to influence and destroy people physically, physiologically, and psychologically, even economically. Cancer is a condition in which any type of body cell turn into abnormal cell. The range of abnormality cannot be controlled as it involves abnormality in DNA manipulation, functional abnormalities and metabolic abnormalities (Stewart., 2014). Hence, currently cancer can be considered as one of the leading global threats to humans. Now, we are in an era to know at the core level of this abnormality and also to generate personalized therapy without side effects.

Plants hold the credit of being used and investigated for therapeutic purposes in all age of human life (Uma Dvei et al., 2013).

Many of the Indian medicinal plants are also widely investigated (Paolo Scartezzini., 2000). An important difference between the traditional medicines compared with the modern medicines is, mostly crude preparations were used as remedial formulation in traditional treatment methods. But, the modern medicines keenly focus on the specific active principle with its definite mechanism of action.

Considering this fact, we bring into play a traditionally used valuable medicinal plant Indigofera aspalathoids, commonly known as Sivanar vembu in Tamil. Previously, we reported the cytotoxic and apoptotic efficacy of silver nanoparticles synthesized from I. aspalathoids (Krishnasamy et al., 2014). In a CSIR publication, 2001., there an citation which mentions about the antitumor proprety of stem of Indigofera aspalathoids. The antitumor efficacy of the ethanol extract of the pant I. aspalothoides on the Ehrlich Ascites Carcinoma mice model was reported (Rajkapoor et al., 2004). The anticancer activity of methanol extract was also reported in Swiss Albino mice (Gupta et al., 2007). The hepatoprotective activity of methanolic extract of I. aspalathoides exhibited hepatoprotective activity (Gupta et al., 2004). Now, in our current research, we report the effect of Colchicine one of the active principle isolated from I. aspalathoides into action against Hepatocellular carcinoma; Hep3B cell lines. Colchicine is important alkaloid, which is widely used as an important pharmaceutical compound for the treatment of cancer as an antimitotic agent (Sivakumar et al., 2004 and Bhushan et al., 2004).

MATERIALS AND METHODS:

Compound Isolation: Plant leaves of Indigofera aspalathoids were collected from a village near Trichy, Tamil Nadu. India. The leaves were shade dried, semi crushed and subjected for Soxhlet extraction using ethanol. The extract was collected, concentrated using rotary evaporator. The compound was isolated using hexane, chloroform and methanol in specific ratio by column chromatography (Unpublished data).

Maintenance of Cell line:

Hep3B cell line (ATTC) was purchased from NCCS, Pune. The cells were grown in T-culture flask containing MEM medium supplemented with 10% FBS. The cells were detached using Trypsin-EDTA solution, centrifuged at 1000 rpm for 5 minutes. The pellet was resuspended using MEM with 10% FBS and used for further analysis.

Cell Proliferation Assay:

MTT assay was done to check the proliferation of Hep3B cell lines (Safadi et al., 2003). Cells were placed in a 24 well plate (5X10⁴ Cells/Well) and incubated at 37° C after washing twice with serum free medium. The cells were then treated with different concentrations of colchicine for 24 hours. Then, 500μl of MTT solution with MEM (0.5 mg/ml) was added to all the wells and kept for 4 hrs in the CO₂ incubator. The medium was discarded and the cells were again washed and resuspended with 1ml of 1X PBS and 500 µl of solublizing solution. The sample was measured at 620nm. The OD values of control and sample were taken and the graph was plotted.

Acridine Orange/Ethidium Bromide (AO/EB) staining:

Ethidium bromide and Acridine Orange (100µg/ml both) were added to the cells and kept in dark at room temperature. The morphological changes were observed using a fluorescence microscope (Ribble et al., 2005).

Assessment of Nuclear Morphology by Propidium Iodide Staining:

Propidium Iodide staining was done with reference to Mohan et al., 2010. Cells were plated in 6 well plate (5X10⁴), grown at 37°C in a CO₂ incubator and treated with colchicine for 24 to 48 hours. Apoptosis of cells were observed using fluorescent microscope.

Determination of Apoptotic population using Cell cycle analysis:

To determine the growth inhibition, after propidium iodide treatment the cells were related to cell cycle arrest or apoptosis, the cell cycle distribution was quantiﬁed using ﬂow cytometry. Exponentially growing Hep-3B cells were treated with colchicines for 48 h and then subjected to cell cycle analysis.

RESULTS AND DISCUSSION:

The Colchicine, extracted from Indigofera aspalathoids is widely used in the field of medicines, particularly for the cancer treatment, drug delivery, commercial appliances and sensors (Rajkapoor et al., 2007). According to Rajkapoor et al., 2006, confirmed the hepatic protective property of alcoholic stem extract of Indigofera aspalathoids. Compared to in-vivo studies, in vitro studies benefit from being faster, lower cost, allowing greater control, and minimizing ethical concerns by reducing the number of laboratory animals required for testing. The chloroform extract of I. aspalathoids generated good anti-hepatotoxic activity was reported by Malaya., 2007.

Colchicine is, an alkaloid, mentioned in one of the seven Upanishads in the Indian medicine, which as then used as a treatment cure many ailments like Gout, Famililal Mediterranean fever but may prove fatal on misuse as it is a semi poisonous drug (Jason et al., 2004). Its antibacterial and antimicrobial activity a reported by Rakesh , 2012.

In this current research in vitro cytotoxicity effect of phyto-extracted colchicine from the leave samples was screened for its anticancerous properties using MTT assay. In-vitro cytotoxicity of colchicine from I. aspalathoids was evaluated on Hep3B cell line. The potential effects of colchicine on proliferation and survival of Hep-3B cells were tested by exposing to 0–1000 μg/ml for 72 h. Figure 1, 2 and 3 show that, it can induces cell death in a dose- dependent manner, as determined using MTT assay. The results show that phyto-extract colchicine induces cell death significantly with an average IC₅₀value of 344.25 μg/ml ±4.45 (Graph1).

Fig.1: MTT assay

Microscopic View of cell lines:

The morphological changes of cell lines were observed using phase contrast microscope. Control cell showed typical polygonal and cobblestone monolayer appearance (Fig. 2a). Significant decrease of the number of Hep3B cells treated with colchicine for 24 h was observed compared to the control group (Fig. 2b). Furthermore, the cell treated with colchicine for 48h began to have morphological changes; showing round-shaped cells poorly adhered to the culture flasks (Fig. 2c)

Fig 2a: Control (40X)

Fig 2b: 24 hrs after treatment with IC50 concentration

Fig. 2c: 48hrs after treatment with IC50 concentration

To understand the morphological change of Hep3b cells before and after colchicine treatment, the cells were observed by Acridine Orange/Ethidium Bromide (AO/EB) double staining after treatment at different time points. After 48 hours of incubation, the cells were subjected to staining and green represents viable cells, which are viewed as green fluorescence (Fig 3a), whereas bright greenish yellow fluorescence, which represents early apoptotic cells, and reddish or orange fluorescence represents late apoptotic cells. As shown in (figure 3b), Hep 3B cells after 48 hours of treatment, showed slight changes in cellular morphology, including chromatin condensation and fragmented nuclei.

AO/EB double staining

Fig. 3a: Control (X40)

Fig. 3b: 48hrs after treatment with IC50 concentration

Normal animal cells when stained with acridine orange dye emits yellowish – green fluorescence and during cellular damage the fluorescences hanges to rd olour, due to the drop in the cellular p H (Kronvall and Myher, 1977). In this current research, AO/EB staining of colchiine treated and untreated cells exhibited fluorescence, which confirms that the colchicine treatment lads to Hep3BS cell death.

Detection of Apopotosis by Propidium iodide staining and flow cytometry:

According to Carlo and Ildo, 2006; the carcerous cell after treatment with propidium iodide and after subjecting to flow cytometry the cell cycle can be accurately calculated. The propidium iodide staining method is suitable for the determination of apoptotic morphological characteristics of a Hep 3B cell line, which is further confirmed by a stronger apoptosis signal with increase of time (Fig. 4a control & 4b colchicine treated cells). From the growth studies, it is evident that, the control cells distribution shows about 71.2 % of cells belong to G0/G1 phase, 15.2% of cells were belongs G2/M phase, 6.7% of cells belongs to S phase and finally only 5.66% of the untreated control cells were in the apoptotic phase that is Sub G0/G1 phase (Graph 2). However, upon treatment with colchicines, causing signiﬁcant accumulation of the cells in the apoptotic phase (sub G0/ G1 phase) i.e., about 18.57% of the cells undergo apoptosis. These ﬁndings indicate that treatment with colchicine led to a decrease in the populations of Hep 3B cells i.e. in the G0/G1cells population decreases from 71.2% to 61.42% and finally in G2/M phase from 15.2% to 9.51% with corresponding increase in sub G0/G1 phase. From the cell cycle analysis it is clearly evident that the colchicine induces apoptotic mode of cell death (Graph 3).

Detection of Apoptosis Using PI staining

Fig. 4a. Control (40X)

Fig.4b. 48 hrs after treatment with IC50 concentration

Arrows indicates the cells with fragmented nuclei

The apoptoic poulation was determined using cell cycle analysis.

Graph 2. Control Hep3B cells

Graph.3: Cell cycle analysis of cells treated with colchicine

According to Balasubramanian et al., 2005; the enzymatic and non-enzymatic stress markers were elevated in rat when treated with DEN (D-Nitrosdiethylamine) and on oral treatment with the ethanolic extract of Indigofera aspalathodies, the stress markers level was reduced tremendously. The above mentioned results show that the phyto-extract colchicine inhibits the proliferation of the neoplastic cells. The toxicity of phyto-extract colchicine on Hep3B cancer cell line results demonstrate a concentration-dependent toxicity with 344.25 ±4.45 μg/ml of IC₅₀ proved that the product is more toxic to cancerous cells, comparing to other mode of treatments like heavy metal ions or other synthetic chemotherapy. Hence, we conclude that, this phytobased colchicine can serve as a potential anticancer agent, in a site specific deliverance system.

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Received on 03.03.2016 Modified on 01.04.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 386-390

DOI: 10.5958/0974-360X.2016.00069.X