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Table of Contents
Year : 2022  |  Volume : 6  |  Issue : 2  |  Page : 46-51

Cytotoxic and apoptotic induction activity of protein isolated from blood clam (Anadara (tegillarca) granosa, Linnaeus 1758) Against breast cancer t47d cell line

1 Immunology, Pascasarjana, School of Pascasarjana, University of Airlangga, Indonesia
2 Immunology, Pascasarjana, School of Pascasarjana; Department of Medical Microbiology, Faculty of Dentistry, University of Airlangga, Indonesia
3 Department Animal Physiology, Faculty of Biology, University of Gadjah Mada, Indonesia
4 Immunology, Pascasarjana, School of Pascasarjana; Department of Oral Biology, Faculty of Dentistry, University of Airlangga, Indonesia

Date of Submission20-Oct-2021
Date of Acceptance22-Oct-2021
Date of Web Publication08-Nov-2022

Correspondence Address:
Dr. S Theresia Indah Budhy
Department of Oral Biology, Faculty of Dentistry, University of Airlangga, 60132 Surabaya
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mtsp.mtsp_9_21

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Background: Cancer has become a major cause of death today. These risk factors are also balanced with the development of drugs, one of which comes from nature. Medical products derived from natural resources such as blood clam (Anadara (tegillarca) granosa) have been already developed. Crude protein extracted from blood calm contains a 20-kDa protein which had been known to be able to inhibit the growth of HT-29 cell line. Purpose: This study aimed to determine cytotoxic activity and apoptotic effect of crude protein which is isolated from blood calm against breast cancer T47d cell line. Materials and Methods: This study used the ammonium sulfate precipitation method for the isolation of a crude protein of blood clams. MTT assay method was performed to determine cytotoxicity and to know the value of IC50, apoptosis test qualitatively and quantitatively was done by double-staining using ethidium bromide and acridine orange as well as flow cytometric dye using annexin V dyes and propidium iodide. Findings: Crude protein clams of blood have IC50 value of 11.11μg/ml and can induce apoptosis of breast cancer cell line (T47D) at 15 μg/ml concentration by double-staining method, but the calculation with flow cytometry method still shows different results in this study. Research Limitations: This study observed the inhibitory concentration of crude extract consisting of proteins with various molecular weights against the t47d cell line. Originality: The results of this study indicate that crude protein clams are cytotoxic and can induce apoptosis in breast cancer cell lines (T47D).

Keywords: Anadara (tegillarca) granosa, T47d cell line, MTT assay, double staining, flow cytometry

How to cite this article:
Ardi Putra FR, Setyabudi RJ, Wijayanti N, Indah Budhy S T. Cytotoxic and apoptotic induction activity of protein isolated from blood clam (Anadara (tegillarca) granosa, Linnaeus 1758) Against breast cancer t47d cell line. Matrix Sci Pharma 2022;6:46-51

How to cite this URL:
Ardi Putra FR, Setyabudi RJ, Wijayanti N, Indah Budhy S T. Cytotoxic and apoptotic induction activity of protein isolated from blood clam (Anadara (tegillarca) granosa, Linnaeus 1758) Against breast cancer t47d cell line. Matrix Sci Pharma [serial online] 2022 [cited 2023 Mar 28];6:46-51. Available from: https://www.matrixscipharma.org/text.asp?2022/6/2/46/360584

  Introduction Top

Cancer is the second-leading cause of death in the world. Recently, it has been estimated that 10 million people have died due to cancer. Breast cancer is the highest type of cancer found in women with cancer by the WHO, 2021. The maturation of cancer cells through various processes and involves various types of cells. Therefore, the early detection and elimination of these cells is a promising way to overcome the problem of the spread of cancer cells.[1] Breast cancer can be started from the formation of a tumor in the duct gland which undergoes unlimited division to form a tumor which is then able to develop into cancer.[2] This development can be initiated by mutations that occur in oncogene genes such as human epidermal growth factor 2 (HER2) and estrogen receptors.[3] The mechanism of development and spread of tumors into cancer in breast cells is influenced by a microclimate based on inflammatory events and the avoidance of macrophages. This process triggers the malignancy of cancer cells.[4],[5]

Research on cancer cells has been carried out since 1962 both in vitro and in vivo.[6] In addition to HER2, breast cancer can be caused by a variety of heterogeneous genes. The existence of cell lines can be an alternative in studying the heterogeneity caused by cancer cells.[7]

T47D cell lines are cancer cells isolated from the epithelial tissue of the breast gland of a 54-year-old female patient. This cell has long been used as a model because it is considered ideal for observing the specific effect of a test material.[8]

Chemotherapy has the main goal of killing and stopping the growth of cancer cells directly.[9] However, this therapy is considered to have a fairly damaging effect on healthy cells (not permanently). Therefore, it is very necessary to find another alternative to this treatment.[10]

At present, almost 50% of discoveries in the field of medicine come from natural ingredients and it is estimated that two out of three anticancer drugs are of natural origin.[11] Protein extracts from blood clams are known to induce apoptosis in various cells such as hepatocellular carcinoma cell line (BEL-7402 cell line) and[12] human ovarian cancer cell lines SKOV-3 and OVCAR-3.[13]

This potential is proven by the discovery of bioactive components from Tegillarca granosa including fur and compound (antimicrobial), palmitic acid (antioxidant), fatty acid ester (antidiabetic, antiasthmatic, and anticancer), phenolic compound (analgesic), and plasticizer compound (antimicrobial).[14] Anadara protein (Tegillarca) granosa has a potential molecular weight as an anticancer between 15 and 23 kDa.[13]

In Indonesia, Anadara (Tegillarca) granosa Linnaeus, 1758, is a consumable clam with the regional name blood clam. This clam has a very wide distribution in the Indo-Pacific waters. According to Narasimham, the shellfish has an ideal habitat in a fine muddy intertidal zone with low salinity and protected from waves.[15]

The benefit of this study is to provide information about the potential of blood clams (Anadara (Tegillarca) granosa) crude protein as a potential anticancer drug candidate by observing its cytotoxic and apoptotic activity against breast cancer cell lines.

  Materials and Methods Top

Crude preparation of protein extract

Samples of Anadara (Tegillarca) granosa were collected from the Depok beach fish auction place, Bantul, Yogyakarta, and then identified using the Food and Agriculture Organization (FAO) Species Identification Guide for Fishery Purpose, World Mollusc Species Database, compendium of seashells, and verification of species names using World Register of Marine Species (WoRMS).

Two hundred grams of Anadara (Tegillarca) granosa visceral mass was washed with aquades. The sample was crushed and then dissolved in Phosphate-buffered saline (PBS) (0.03 M, pH 8.0). The sample in PBS was then filtered using a funnel that had been coated using Whatman No 2 filter paper in a cool box which has been added with an ice pack to keep the temperature constant in that sample then centrifuged at 8000 rpm for 30 min at room temperature. The supernatant was then taken and added with ammonium sulfate solution with a percentage of 36.47 g/100 ml of sample in PBS to increase its concentration. The sample which has been added with ammonium sulfate is then deposited for one night. The precipitation results were then centrifuged at 8000 rpm for 30 min two times to separate the crude protein from the remaining ammonium sulfate. Crude protein is then stored.[16]

Cytotoxicity test

The t47D cell culture was obtained from the Universitas Gadjah Mada (UGM) Integrated Research and Testing Laboratory (LPPT). Cells were cultured by LPPT in complete media (RPMI 1640 with 10% fetal bovine serum (FBS) + pen-Strep + fungizone). The t47D cells that were ready for harvest were then transferred to a 96-well microplate with a density of 1 × cells/100 μl/well with the following test design: (1). negative control without treatment; (2). solvent control using PBS; (3). solvent control using + ammonium sulfate; and (4). the concentration treatment group was 1,25; 2,5; 5; 150,625; 25; 30,125; 50; 125; 250 and 500 μg/ml.

T47D cell cultures were incubated at 37°C, at 5% for 24 h. Cytotoxicity test was carried out by administering MTT (0.5 mg/mL) 100 μL. The cell suspension was incubated at 5% temperature for 4 h. The reaction was stopped by adding a 10% Safety Data Sheet (SDS) stopper in 0.1 N Hydrochloric Acid (HCL) at 100 μl/well. The cell culture that has been added with a stopper is then left overnight to see the optimum color change. The absorbance value with a wavelength of 550 nm.[17]

Apoptotic test

Double staining

The double staining method was used in the apoptosis test using ethidium bromide–acridine orange staining. The dye solution was mixed with a ratio of ethidium bromide–acridine orange (10:3). A total of 1 mg ethidium bromide are mixed with 0.3 mg of acridine orange and then dissolved in 1 ml of sterile PBS.

Flow cytometry

Apoptosis testing used t47D cells with a density of 250 cells/well which were cultured in a 24-well microplate. The cells were then incubated in a CO2 incubator for 24 h so that the cells adhered. After 24 h of incubation, the cell medium was replaced with a treatment medium and incubated again for 24 h to see the effect of the extract treatment on t47D cells. After treatment, cells were incubated for 24 h, and t47D cells were then harvested and dissolved in PBS. Cells in PBS suspension were then centrifuged at 1000 rpm for 10 min. The supernatant was then discarded and the cell pellet was suspended using a binding buffer of 500 μl, centrifuged at 1000 rpm for 10 min. The centrifuged supernatant was discarded, and the pellet obtained was then added with 2 μl Annexin V-FITC and 2 μl of propidium iodide (PI) then incubated for 10 min at room temperature in the dark. The suspensions were then read using flow cytometry.[18]

Calculation of the value of apoptosis using double staining is calculated using the formula.[19] Counting was carried out for at least 100 cells and repeated for three replications so that the total cells observed for each treatment were 300 cells.[20] Apoptotic values using the flow cytometry method will be divided into four quadrants, namely, the lower left quadrant (live cells), lower right (apoptotic early cells), upper right (apoptotic cells), and upper left (necrotizing cells).

  Results and Discussion Top

Anadara (Tegillarca) granosa has not been evaluated by the International Union for Conservation of Nature and Natural Resources.

Protein precipitation using ammonium sulfate of 70%, 80%, and 90% gave the results in the form of protein with concentrations of 780, 733, and 550 μg/ml. protein precipitation with ammonium sulfate with a percentage of <20% is used for purification of multi proteins with large molecular weights, whereas protein precipitation using ammonium sulfate with a percentage of >55% can be used for protein precipitation with small protein weights (Wingfield, 2001) as shown in [Figure 1].[21]
Figure 1: Morphology of Anadara (Tegillarca) granosa (A) Ribs. (B) Former visceral mass attachment muscles. (C) Dorsal margin. (D) Interior valve and the concentration of crude protein as a result of precipitation with ammonium sulfate

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T47D cell cultures were grown in RPMI-1640 medium with the addition of FBS as a growth factor and penicillin-streptomycin which functions as an antibiotic and fungizone which functions as an antifungal. The cells that were ready to harvest were characterized by the formation of monolayer formation with confluency of >80% [Figure 2]. T47D cells have a doubling time of 32 h.[22]
Figure 2: Observation of apoptosis of T47D cells using a fluorescence microscope (a) Control cells. (b) Doxo control. (c) Concentration of 10 μg/ml (d) Concentration of 15 μg/ml (e) Concentration of 20 μg/ml) magnification 10 x 40

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Culture cytotoxicity test aims to evaluate the toxic properties of a material or extract against t47D cells treatment of crude protein using a concentration series of 1,25; 2,5; 5; 150,625; 25; 30,125; 50; 125; 250 and 500 μg/ml aimed to determine the level of cytotoxicity of the treatment against t47D cells [Figure 3].
Figure 3: T47D cell density at the 90% confluency (left) and percentage of t47D cell viability against crude protein concentration log

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[Figure 3] T47D cell viability formed a sigmoid curve concerning the concentration log. The higher the concentration used is directly proportional to the amount of cell death. In the graph, it is known that the IC50 value obtained is 11,11 μg/ml with an R2 = 0.9098.

According to Weerapreeyakul et al. (2012) explained that the cytotoxicity effect of the extract on cells is divided into three, namely, very strong cytotoxicity with an IC50 value of <10 μg/ml, strong cytotoxicity with an IC50 value of between 10 and 100 μg/ml, and moderate cytotoxicity with an IC50 value of 100–500 μg/ml.[23] Merghoub et al., in the American National Cancer Institute, states that the effect of the extract has the potential as an anticancer drug if it has an IC50 value of <30 μg/ml.[24] Crude protein Anadara (Tegillarca) granosa has an IC50 value of 11,11 μg/ml so it can be categorized as having a strong cytotoxicity value and potential as an anticancer drug.

Cell death caused by the test compound can be observed by double staining, staining technique using two kinds of dyes, namely, AO and EB (acridine orange and ethidium) chosen because they can be used to observe live cell differences.[25],[26]

Observations under a fluorescence microscope as in [Figure 2] show that ethidium bromide dye will show a green color, whereas acridine orange will form an orange color under a fluorescence microscope.[27],[28],[29]

Crude protein treatments with a concentration series of 10, 15, and 20 μg/ml show that there is no bond between cells so the cells appear separated (unlike control cells). The treatment of 10 and 15 μg/ml [Figure 4] showed that many cells experienced early apoptosis (early apoptosis) with marked cells still green but pyknosis occurred in the cell nucleus. In addition, cells that underwent late apoptosis and necrosis were also found, marked by cells and nuclei that were already orange. In the treatment of 20 μg/ml [Figure 4], no living cells were found which was characterized by the absence of green luminescence, this concentration was a concentration >IC50.
Figure 4: Apoptosis using flow cytometry method: control cells (negative control), doxorubicin (positive control), treatment 1 (10 μg/ml), treatment 2 (15 μg/ml), and treatment 3 (20 μg/ml)

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Treatment of 10 μg/ml induced apoptosis of T47D cells by 44%. Treatment of 15 μg/ml induced apoptosis of T47D cells by 60%. Treatment induced apoptosis 20 μg/ml T47D cells by 100%. Based on the graph of the ratio of dose concentration to the percentage of apoptosis in T47D cells [Figure 5], it is known that the induction of apoptosis in T47D cells is influenced by the concentration of crude protein extract Anadara (Tegilarca) granosa. Research by Li et al. using K562-induced mice showed that caspase-3 expression increased in line with increasing doses of blood clam protein.[30]
Figure 5: Percentage of T47D cell apoptosis against crude protein extract Anadara (Tegillarca) granosa

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Quantitative measurement of apoptosis can be done using the flow cytometry method. This technique uses two dyes, namely, annexin V which will bind phosphatidylserine on the surface of cells undergoing apoptosis and PI is a dye used to identify cell death in a population.[31],[32]

The design of the apoptotic treatment used untreated T47D cells as a negative control, doxorubicin as a positive control, and three series of extract concentrations, which are 10, 15, and 20 μg/ml. The highest percentage of apoptosis was found in treatment 2, which is 8.13%. The apoptotic results obtained were not affected by the percentage of protein extract, the concentration of 15 μg/ml had a higher apoptotic value than the concentration of 20 μg/ml. The results obtained are in line with previous research examined that higher protein concentrations in Anadara (Tegillarca) granosa had a lower cytotoxic effect.

  Conclusion Top

Apoptosis is a program of cell death that usually occurs in the stage of cell development as a mechanism for the hemostasis of these cells. Apoptosis is also a cell defense mechanism against disease or harmful agents. Some many conditions and stimuli can cause cells to undergo apoptosis, including physiological factors and pathological factors.[33]

Crude protein Anadara (Tegillarca) granosa contains proteins with various molecular weights that have potential as an antitumor. Shuangshuang et al. (2015) explained that a protein with a molecular weight of 20 kDa has great potential as an antitumor.[17] These proteins can inhibit the proliferation of HT-29, HepG2, HeLa, and A549 cells in vitro. Anadara (Tegillarca) granosa protein has potent cytotoxic and apoptotic activity. Xu et al. (2014) explained that Anadara (Tegillarca) granosa protein inhibits the growth of cancer cells and causes cells to be trapped in the G0/G1 phase. Xu (2014) further explained that Anadara (Tegillarca) granosa can induce apoptosis by suppressing the synthesis of Ki67. In addition, Anadara (Tegillarca) granosa extract also had a positive effect on increasing the expression of caspase-3. Increased expression of the caspase-3 gene and inhibition of Ki67 synthesis causes cells to undergo apoptosis.

The mechanism of apoptosis can occur from various pathways, namely, the intrinsic pathways, the perforin pathway is affected by radiation, toxins, hypoxia, and others. This pathway will induce caspase-9 which will activate caspase-3 as the executor. The perforin pathway is influenced by cytotoxic T-cells which activate caspase-10 and then activate caspase-3 as the executor. The extrinsic pathway is influenced by death ligands and death receptors. The extrinsic pathway is characterized by the formation of caspase-8 which will then activate caspase-3 as an executor. Other studies explained in their research using BEL-7402 cells that the crude protein Anadara (Tegillarca) granosa induces apoptosis through the extrinsic pathway by overexpressing caspase-8 and decreasing pro-caspase-8 expression. Caspase-8 expression increased with increasing dose of Anadara (Tegillarca) granosa protein concentration and increased caspase-8 expression level was also followed by an increase in caspase-3.

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Conflicts of interest

There are no conflicts of interest.

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