Effects of α, β momorcharin extract of momordica charantia in intracellular free calcium on cancer cell lines

Abstract

The Momordica charantia L., (family: Cucurbitaceae) is a scientific name of the plant and its fruit. It is also known by other names, for instance in the USA it is known as Bitter gourd or balsam pear while its referred to as the African cucumber in many African countries. This study was specifically designed to investigate the cellular mechanisms whereby alpha, beta momorcharin an extract of M. charantiacan induce cell death measuring the elevation in intracellular free calcium concentrations in three different cancer cell lines 1321N1, Gos-3 and U-87. The results show that incubation of the three cancer cell lines 1321N1, Gos-3 and U-87 with α, β momorcharin can result in significant (p < 0.05) time-dependent increases in [Ca²⁺]_i in all three cancer cell lines compared to control (untreated) cells. Maximal increases in [Ca²⁺]_i was attained after 420 min of incubation. In control (untreated cell lines), [Ca²⁺]_i remained more or less stable in both cell lines after 420 min. The results also show that the increase in [Ca²⁺]_i in Gos-3 cell line was much more pronounced following incubation with α, β momorcharin compared to 1321N1 and U-87 cell line. The results show that incubation of the three cancer cell lines with momorcharin can result in significant (p < 0.05) time-dependent increases in [Ca²⁺]_i in all three cancer cell lines compared to control (untreated) cells. Maximal increases in [Ca²⁺]_i was attained after 420 min of incubation. In control (untreated cell lines), [Ca²⁺]_i remained more or less stable in all three cell lines after 420 min. These results clearly show that α, β momorcharin extract of M. charantia is exerting its anti- cancer effect via an insult to the mitochondria resulting in apoptosis, calcium overloading and subsequently, cell death.

Keywords: Cancer cells; Extract of M. charantia (α, β) alpha; Beta momorcharin; Intra cellular calcium; Cell viability

Introduction

Calcium is the most important physiological cation in cellular regulation [1, 2]. It is the trigger, the promoter and the regulator and moreover, a ubiquitous intracellu- lar signaling molecule which controls a wide range of cellular processes including secretion, membrane transport, contraction, cell proliferation, gene transcrip- tion and even cell death [3-5]. In un-stimulated pancre- atic acinar cells, as well as in other cells, the free intra- cellular calcium concentration [Ca²⁺]_i is between 50– 100 nM. In order to maintain this low resting level, a variety of pumps and uptake systems are present in the plasma membrane and in intracellular organelles to buffer (Ca²⁺) [6]. Calcium mobilisation is dependent upon intracellular calcium stores as well as extracellular calcium medium.

The intracellular level of [Ca²⁺]i in resting cells is main- tained within a normal range of 50 -100 nM. Ca²⁺ home- ostasis is tightly controlled to prevent and overcome problems of cytotoxicity due to its very low diffusibility in the cytoplasm. Distributed throughout the cytoplasm is an extensive array of Ca²⁺ pumps [7, 8]. Many studies have demonstrated mitochondrial Ca²⁺ overload as the link between complement deposition and the observed changes in mitochondrial physiology and the triggering of programmed cell death (PCD) [9]. The mitochondrial Ca²⁺ overload is responsible for the increased O(2)(*-) production [10]. If cytosolic Ca²⁺ rise is not accompa- nied by the accumulation of the cation in the mitochon- drion and consequent production of O(2)(*-), cells usu- ally die by necrosis instead of PCD. For example, mem- brane attack complex assembly (MACA) on the parasit in cell surface allows Ca²⁺ entry in the cell and its accu- mulation in the mitochondrion can lead to O2 produc- tion. This in turn constitutes a PCD signal [11]. In the light of its different potential medicinal values and prop- erties of M. charantia, this study was designed specifi- cally to measure intracellular free calcium on four dif- ferent cancer cell lines by the effects of α, β momor- charin extract of Momordica charantia.

Material and Methodology

Extraction method for either of α or β momorcharin: The whole fruit of bitter gourd was ground and homoge- nized in 2 mM sodium phosphate buffer, pH 7.5. The resulting slurry was then stirred for 3 hrs. to extract the crude proteins. The insoluble component from crude proteins was removed by the filtration and centrifuga- tion at 30,000 x g for 1 hour at 48°C. By using 2 mM sodium phosphate buffer, pH 7.5, the crude protein so- lution was dialysed. The dialysed protein sample was applied to DEAE Sepharose column equilibrated with 2

mM sodium phosphate buffer at pH 7.5. The unbound proteins were then applied to Mono-S column which was equilibrated by 2 mM sodium phosphate buffer at pH 7.5 and eluted by 0.5 m of NaCl. The fraction corre- sponding to either alpha and beta or alpha, beta momor- charin, which was confirmed the N-glycoside activity RNA, was concentrated and dialysed against 20 mM Tris-HCl buffer, pH 7.8. The chromatography was per- formed on Bio Logic DuoFlow system (BioRad, Hercu- les, CA) at 48°C. The purity of α and β momorcharin was examined by SDS-PAGE and gel filtration chroma- tography. The concentration of alpha momorcharin was determined by spectrophotometrically using optical ab- sorbance at 280nm.

Passaging of the Cancer cell lines and Control cell line The culture medium, phosphate buffer solution (PBS), and trypsin (sterile) were removed from the fridge at 4° C and subsequently placed in the water bath at 37°C for 30 min in order to equilibrate. The Laminar flow hood was turned on for 15 min, prior to start of the experi- ment, in order to purge the air inside the cabinet and to reach the maximum cleanliness.

The three different cancer cell lines 1321N1, Gos-3 and U-87 were incubated at 37°C incubator in an atmos- phere of 5% CO2 in air. The cells were examined under the inverted contrast microscope to note the both conflu- ence and general health of the cells. The flask was pas- saged when the cells had reached 70-80% confluence. The medium was aspirated from the cultured flask and was washed with sterile PBS (5 ml if 75 cm2 flask and 2 ml if 25 cm2 flask) in order to remove any traces of serum from the cells. This prevented the serum from inactivating the trypsin which was used to detach adher- ent cells from the cell clump. Trypsin solution (2 ml if 75 cm2 flask or 1 ml if 25 cm2 flask) was pipetted in the flask and incubated at 37°C in an incubator in an atmosphere of 5% CO2 in air for 3-5 min until the cells began to detach. The detachment was confirmed by ob- serving at intervals under an inverted microscope. The cells were left in the trypsin solution for the correct length of time. If the cells were left for a longer period of time, then this would lead to damage of the cells. A volume of 3 ml complete growth medium was then add- ed to the flask to inactivate the trypsin and the cells were pipetted up and down to break up any large cell aggregates. The cell suspension was transferred from flask into 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 min. Following centrifugation, the supernatant was aspirated and the cells were pellet at the bottom of the centrifuge tube. Based upon the cell pellet density volumes of 1 ml to 3 ml fresh medium were suspended in the centrifuge tube. The cell pellet was flicked properly in the medium containing 20 μl of trypsinised cell suspension and 80 μl of tryphan blue (used to detect dead cells in the cell suspension 1:5 ratio). The contents were mixed well together and a haemocytometer test was performed using 1 ml of cell suspensions. This pro- cess helped to assess the total number of the cell suspen- sion present in the centrifuge tube and which was re- quired to make 1 or 2 flasks and to do 96 well plates. Thereafter, the cells were frozen in liquid nitrogen de- pending on the number of cells present per ml. The cell suspension was divided in either one or several flasks (depending on the cell density) and fresh growth medi- um (10 ml to 12 ml if 75 cm² flask and 5 ml if 25 cm² flask) was added to the flasks. These were then placed

Measurement of [Ca²⁺]_i was performed as previously described in the literature [12]. The measurement of [Ca²⁺]_i was performed by seeding a density of 10,000 cells/well in black 96 well plates (Griner, UK) in 100 µl of growth medium. At sub confluence, the cell culture medium was then replaced with FBS free medium for

24 hr. in order to synchronize cells into a non- proliferation stage. The cell were then loaded with 25 µM Fura-2 acetoxylmethyl ester (AM) in cell medium at 37°C for 30 min followed by 30 min at temperature to minimize dye leakage and sequestration into intracellu- lar organelles. After loading, the cells were washed 2-3 times with 200 µl of Hank’s Buffered Salt Solution (HBSS) to remove excess fluorescent dye. The cells were then treated with extract of M. charantiaα, β momorcharin with concentration of 800 µM for differ- ent durations (0 min - 420 min) in a calcium free medi- um. The cells were washed twice with 200 µl HBSS and then a volume of 100 µl HBSS /wells was added to each well. The same procedure was carried out for the un- treated cells. The intensity of the fluorescence’s of Fura- 2 AM was measured at emission wavelengths of 340 nm. The relative changes in calcium concentration using Fura-2 AM were determined by calculations of;

[Ca ²⁺]_i = Kd β (f – f min) / (f max – f).

Kd, the Ca-Fura-2 dissociation constant, β the fluores- cence intensity ratio, excited at 380 nm without and with Ca; f min and f max [13]. All values were ex- pressed as ratio units of the Fura-2 AM fluorescent in- tensity.

Statistical analysis: All control and test data collected from the different experiments were analysed using Sta- tistical Package for Social Sciences (SPSS) version 17, Student’s – t test and ANOVA test. Data obtained were expressed as mean ± standard deviation (S.D). Each experiment was repeated for 4-6 times in duplicate (6 for cell viability and 4 for cell signaling) to ensure the accuracy of results. A value of (p < 0.05) was taken as significant.

Results

Figure 1 and 2 shows the time-course changes in [Ca²⁺]_i expressed as fluorescence ratio units, in (A) 1321N1 and (B) Gos-3 cancer cell lines (solid diamonds) follow- ing incubation with 800 µM of α, β momorcharin for 420 min. The changes in [Ca²⁺]_i in untreated (control) 1321N1 and Gos-3 cancer cell lines (solid squares) are also shown in the figure for comparison. The results show that incubation of the two cancer cell lines with α, β momorcharin can result in significant (p < 0.05) time- dependent increases in [Ca²⁺]_i in both cancer cell lines compared to control (untreated) cells. Maximal increas- es in [Ca²⁺]_i was attained after 420 min of incubation. In control (untreated cell lines), [Ca²⁺]_i remained more or less stable in both cell lines after 420 min. The re- sults also show that the increase in [Ca²⁺]_i in Gos-3 cell line was much more pronounced following incubation with α, β momorcharin compared to 1321N1 cell line.

Figure 3 shows the time-course changes [Ca²⁺]_i in ex- pressed as fluorescence ratio units (A) U87- MG cancer cell lines following incubation with 800 µM of α, β momorcharin (solid diamonds). The changes in [Ca²⁺]_i in untreated (control) U87- MG cancer cell lines are also shown in the figure for comparison (solid squares). in a 5% CO2 incubation.

Measurement of intracellular free calcium [Ca2+]i.

In contrast, with untreated U87–MG cell line basal [Ca²⁺]_i remained more or less the same over 420 min. However, in U87-MG cell line treated with α, β momor- charin [Ca²⁺]_i decreased slightly for 0 - 420 min com- pared to untreated control but then increase gradually for 360 min to 420 min when it was significant (p < 0.05) compared to control untreated cells.

Figure 1. Time-course changes in [Ca²⁺]_i expressed as fluorescence ratio units (intensity) in Fura-2 AM loaded 1321N1 cell lines

Figure 2. Time-course changes in [Ca²⁺]_i expressed as fluorescence ratio units (intensity) in Fura -2 AM load- ed U87-MG cell lines.

Figure 3. Time-course changes in [Ca2+]i expressed as fluorescence ratio units (intensity) in Fura -2 AM load-ed U87-MG cell lines.

Discussion

The results of this study have demonstrated significant anti-cancer effects of alpha, beta momorcharin of M. charantiaon the three different cancer cell lines com- pared to untreated control. Anti-cancer drugs are be- lieved to exert their ‘killing’ effects on cells via differ- ent cellular and sub-cellular mechanisms including dam- ages to the mitochondria and microtubules, inhibition of kinases or by cellular calcium over-load [14-17]. This study employed extract of M. charantiaalpha, beta momorcharin may exert their anti- cancer effects on cell death, namely, apoptosis and cellular calcium homeosta- sis.

The results presented in this study have shown that puri- fied protein of M. charantia, namely alpha, beta momorcharin can elicit marked and significant changes in the activities of intracellular free calcium concentra- tions in all three cell lines employed in this study com- pared to control untreated cell lines.

Apoptosis is programmed cell death and it is associated with damage of cell mitochondria in the body to elevate such intra-cellular mediators such as caspase-3 and caspase-9 and the release of cytochrome-c [18,19]. In previous studies, it was shown that anti-cancer drugs exert their lethality by inducing apoptosis in tumour cells in vitro and in vivo targeting both the mitochondri- al and death receptor pathways [20,21]. There are two major apoptotic pathways in mammalian cells namely the receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway [22]. The receptor-mediated path- way is triggered by activation of cell death receptors followed by the activation of caspase-8, which in turn cleaves and activates downstream caspase-3 [23]. The mitochondrial pathway is initiated by cytochrome-c release from the mitochondria which promotes the acti- vation of caspase-9 through activated caspase-9 which is responsible for the activation of cell death proteases [24, 25].

The results of the present study have shown that alpha, beta momorcharin can evoke significant and time-dependent increases in [Ca2+]i in all the three cancer time cell lines. Their effect was less pronounced in U87-MG cell line compared to others. The question where now arises is: What is the significance of elevated [Ca²⁺]_i in these cancer cell lines following treatment with alpha, beta momorcharin. Previous studies have shown that sustained Ca²⁺ elevation can act as trigger for apoptosis or cell death [13]. In addition, Ca²⁺ overloading in mito- chondria can induce a cell suicide programme by stimu- lating the release of apoptosis promoting factor like the release of cytochrome-c19,20. Cytosolic Ca²⁺ homeosta- sis in resting cells is achieved by balancing the leak of Ca²⁺ (entering from the outside of from the stores) by the constant removal of Ca²⁺ using pumps either on the plasma membrane or on the internal stores. These pumps ensure that cytoplasmic [Ca²⁺]_i remains low and that the stores are loaded with signal Ca²⁺. In most cells, it is the internal stores (eg. ER, SR, mitochondria) which provide most of the signal calcium [7]. Further- more, mitochondria have been found to play a pivotal role in Ca²⁺ signaling [13]. The cellular free Ca²⁺ is an important physiological mediator and regulator in the stimulus-secreting coupling process in different epithelial cells [22], Many studies, have demonstrated mito- chondrial Ca²⁺ overload as the link between comple- ment deposition and the observed changes in mitochon- drial physiology and the triggering of programmed cell death (PCD)6. The mitochondrial Ca²⁺ overload is re- sponsible for the increased O(2)(*-) production8. The rates of mitochondrial membrane potential dissipation and mitochondrial Ca²⁺ uptake may determine cellular sensitivity to Ca²⁺ toxicity under pathological condi- tions, including ischemic injury [14]. It has also been suggested that only a subpopulation of mitochondria undergoes a permeability transition and releases apopto- gens, whereas the remaining, undamaged mitochondria respire normally and produce ATP [21, 22].

The result presented in this study have demonstrated clearly that the anti-cancer effects of alpha, beta momorcharin are mediated via apoptosis and cellular Ca²⁺ overloading.

Conclusion

The results of this study have clearly demonstrated that the isolated and purified protein of M. charantia, name- ly alpha, beta momorcharin can evoke significant de- creases in cancer cell viability (cell death) by exerting their anti-cancer effect on cells via damage of cell mito- chondria body resulting in elevation in elevation of in- tracellular free calcium concentration [Ca²⁺]_i. A combi- nation of all these factors may lead to Ca²⁺ overloading in the mitochondria resulting in cancer cell death. How- ever, further experiments are required to investigate the sub- cellular mechanisms associated with cell death including the involvement of kinase and gene expres- sions for apoptotic mediators.

Conflict of Interests: The author has not declared any conflict of interest.

Acknowledgement: The author highly thankful to University of Central Lancashire, UK, for providing facilities need for this research work.

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