INTRODUCTION:

Breast cancer has been widely recognized as the most deadly cancer. Geographically, Western Europe, and the United States rank highest, followed by South America and Eastern Europe¹. While Asia is relatively lower but still shows the highest incidence of all types of cancer in women. Indonesia itself shows the second-highest incidence after uterine cancer².

Until now chemotherapy is still the main choice in overcoming breast cancer with one of the most widely known uses being doxorubicin^3,4. The use of high doses of doxorubicin and long-term risk of causing cardiotoxicity that can cause death, liver failure, myeloid Ysplasia, and leukemia^5,6,7. Doxorubicin-based therapy also triggers chemotherapy resistance⁸ through activation of Akt⁹ and NFkB¹⁰.

Efforts to increase the response of therapy and minimize side effects, toxicity to chemotherapy resistance continue to be developed. One such development strategy is the use of chemopreventive agents which are non-toxic or non-toxic compounds derived from natural substances as a combination of chemotherapeutic agents known as co-chemotherapy¹¹.The purpose of using this chemopreventive compound is directed at increasing the sensitivity of chemotherapy agents to cancer cells, conversely, normal cells can reduce the toxicity and side effects of chemotherapy agents.

Deoxyelephantopin is one of the interesting natural compounds to be developed as a chemopreventive agent, which until now there has been no report related to its use as a chemotherapy agent. This compound belongs to the sesquiterpene lactone group derived from the plant Elephantopus Scaber¹². The anticancer activity of this compound has been demonstrated on several cancer cell cultures of SMMC7721, Hela, KBM-5, T47D, A549 and CaCO₂^{13,14,15,16,17}. In KBM-5 myeloid leukemia cancer cell culture, this compound can suppress NFkB¹⁸. In T47D breast cancer, culture cells, and A549 lung cancer culture cells this compound can trigger apoptosis through caspase-3 activation and cell cycle arrest. Explanation of cell death through apoptosis has been more clearly reported Kabeer, et al.,¹³ through suppression of PI3K, Akt and mTOR, conversely an increase in ROS levels and activation of pro-apoptotic proteins (Bad, Bax) and protein caspase (9,8,3), activates the β-Cathenin pathway by suppressing the expression of c-myc and cyclin D1 thus triggering apoptosis.

This potential compound as a chemotherapy combination agent related to anticancer ability in many of the cancer cells model above, molecularly this compound has also been reported to suppress NFκβ a gene that has been shown to broadly trigger cell proliferation activity and suppress apoptosis¹⁸. In addition to normal cells, deoxyelephantopin is reported to be non-toxic as a treatment of blood lymphocyte cells¹⁹.

This study aimed to determine the extent to which the role of deoxyelephantopin increases doxorubicin sensitivity in triggering apoptosis in MCF-7 breast cancer cell culture cultures.

MATERIALS AND METHODS:

Research Materials:

Deoxyelephantopin (FitoPure®, Catalog No. RC051301) contains ≥ 98% pure HPLC. Deoxyelephantopin is dissolved in Dimethyl Sulfoxide (DMSO) (Sigma). MCF-7 breast cancer cells come from the collection of Cancer Chemoprevention Research Center, Faculty of Pharmacy, Gajah Mada University. Cell culture was grown in Dulbecco's modified Eagle's medium (DMEM) culture medium containing 10% (v/v) (Gib /) Foetal Bovine Serum (Gibco), penicillin-streptomycin 1% (v/v) (Gibco). Trypsin-EDTA 0.25% is used to help the release of cells attached to the flask. Cell death is determined by the trypan blue (Sigma) staining method.

Research Methods:

Combined Cytotoxic Test Using the MTT Method:

A total of 5x10³ cells/100µL^-1 were distributed into 96 well plates, then incubated for 24 hours in a CO₂ incubator to adapt and adhere to the well. The combination test compounds respectively in the series concentrations of 2/5 IC₅₀, 1/3 IC₅₀, 1/5 IC₅₀, and 1/10 IC₅₀ μg/mL in 100μL of culture media were added to the wells. The combination composition of doxorubicin-deoxyelephantopin is determined as follows:

At the end of the incubation period, the medium was removed and 100µL PBS was added per well for washing. MTT 5 mg.mL^-1 in 100µL of culture media was added to each well and then incubated for 3 hours at 37°C. The MTT was discarded, then the SDS stopper was added to dissolve formazan salt. Cells were incubated for 24 hours at room temperature and protected from light. At the end of the incubation, the plate shook using a shaker for 15 minutes, then read with ELISA Reader at λ = 595nm. The data obtained in the form of absorbance of each well then converted into a percentage of viability through the following equation:

Abs of treatment cell – Abs of control media

Viability=------------------------------------------------------X 100%

Abs of control cell – Abs of control media (1)

The magnitude of the concentration of each combination compound is obtained from the combination viability data through a single regression equation for each of these compounds. The single doxorubicin regression equation is y = -0.05x 72.40 (r = 0.946 with IC₅₀ 448nM), and the single deoxyelephantopin regression equation is y = -2.985 + 83.94 (r = 0.956 with IC₅₀ 11.2 µg/mL). The two single regression equations were obtained from processing a single viability data for each of doxorubicin and deoxyelephantopin compounds that the authors have reported in another journal, but have not yet been published.

Then the value of the Combination Index (CI) is determined using the equation:

CI = (D)1/(Dx)1 + (D)2/(Dx)…………………………(2)

Where D1 and D2 are the sample concentrations used in combination treatment. (Dx)1 and (Dx)2 are single concentrations that can produce effects as large as those given a combination treatment²⁰. The CI or Combination Index obtained is interpreted as follows: < 0.1 synergist very strong, 0.1-0.3 synergist strong, 0.3-0.7 synergistic, 0.7-0.9 mild-moderate synergistic, 0.9-1.1 mild-moderate antagonist, 1.45-.3 antagonist, > 3 strong-very strong antagonists.

Cell Death Test:

Cells were planted 5x10⁵ in each six-well plate well. After all, cells are attached to the base plate, the medium is removed and washed using PBS. Enter the test substance by following a predetermined concentration of 1ml into each well then incubated 24 hours in a CO₂ incubator. Cells are taken back from the incubator and the medium is removed, then washed with PBS of 1 ml/well. PBS was removed and put 1ml trypsin into each well. The incubation returns for 5-15 minutes in the CO₂ incubator. If all cells have been released, add 1ml medium/well and transfer into 2ml microtubes, then centrifuge with speed 2000 rpm, 10 minutes. Pellets are taken and the supernatant removed, then add 1ml medium in each tube. Dilute and take 10 ul, then transfer to a 0.5ml centrifuge tube and add 10 ul trypan blue, homogenize. Take 10ul, move it onto the slide, then do cell calculations under a microscope. Blue colored cells are dead cells while clear/white cells are living cells. Cells are calculated based on the following equation,

Total dead cells/mL = average number of dead cells per square x 10⁴…………………………………………(3)

RESULT AND DISCUSSION:

Result:

Combined Cytotoxic Test:

The results. of the viability of these combinations are shown in Table 1 below

Table 1: Comparison of the viability of deoxyelephantopin, doxorubicin, and combinations

		Doxorobucin
		1/10 IC₅₀	1/5 IC₅₀	1/3 IC₅₀	2/5 IC₅₀
Deoxyelephantopin	Viability	79.77 %	70.45 %	68.41 %	64.77 %
1/10 IC₅₀	99.32 %	61.59%	51.36%	42.73%	45.68%
1/5 IC₅₀	87.27 %	48.64%	55%	41.14%	50.91%
1/3 IC₅₀	77.27 %	51.36%	47.04%	45.23%	43.409%
2/5 IC₅₀	63.18 %	46.14%	48.18%	40%	45.68%

Combination Index:

The combination test results obtained by the combination index as in (Figure 1 and Table 2) follows.

Figure 1: Effects of the combination treatment of deoxyelephantopin don with doxorubicin on the viability of cancer cell culture MCF-7

Table 2: Deoxyelephantopin Combination Index (CI) values with doxorubicin on MCF-7 Cancer cell culture

Deoxyelephantopin (µg/mL)	Doxorubicin (nM)
Deoxyelephantopin (µg/mL)	1/10 IC₅₀	1/5 IC₅₀	1/3 IC₅₀	2/5 IC₅₀
1/10 IC₅₀	0.32	0.29*	0.28*	0.39
1/5 IC₅₀	0.25*	0.44	0.34	0.57
1/3 IC₅₀	0.37	0.40	0.46	0.51
2/5 IC₅₀	0.39	0.50	0.46	0.62

Based on the combination viability data, it was found that IC₅₀ doxorubicin was lower than IC₅₀ doxorubicin in a single treatment.

Table 3: Increased sensitivity of doxorubicin when combined with deoxyelephantopin

Deoxyelephantopin (µM)	Viability Dead (% death)	IC₅₀ Doxorubicin (µM)	IC₅₀ Dox + Deoxy (µM)	Enhancement Sensitivity
1/10 IC₅₀	99.32	448	107.68	4.16 x
1/5 IC₅₀	87.27	448	64.72	6.92 x
1/3 IC₅₀	77.27	448	52.13	8.59 x
2/5 IC₅₀	63.18	448	12.46	35.95 x

Table 4: Differences in the number of living / dead cells treated through the trypan blue staining method

Repetition	Number of living and dead cells per treatment
	Doxo IC₅₀		Deoxy IC₅₀		Comb IC₅₀		Doxo 1/2 IC₅₀		Deoxy 1/2 IC₅₀		Comb 1/2 IC₅₀		Control
	Live	Dead	Live	Dead	Live	Dead	Live	Dead	Live	Dead	Live	Dead	Live	Dead
1	408000	64000	360000	32000	200000	72000	312000	24000	420000	40000	300000	60000	592000	32000
2	424000	56000	432000	60000	220000	88000	488000	40000	440000	40000	344000	76000	580000	40000
3	448000	56000	368000	52000	260000	108000	496000	32000	400000	32000	332000	68000	588000	48000
Average	426666	58666	386666	48000	226666	89333	432000	32000	420000	37333	325333	68000	586666	40000

Life/death cell counting through "trypan blue" coloring:

The results of the cytotoxic test were confirmed quantitatively by counting life/dead cells using staining with trypan blue. Living cells with intact membranes will not absorb trypan blue dye, so the cells appear clear. While the dead cells that have damaged the membrane will absorb the trypan blue dye into the cytosol and react with intracellular proteins so that the cells will appear blue. The complete data of living / dead cells as in Table 4 and graphs of differences in the rate of cell death of each treatment as in Figure 2 below.

Figure 2: Graph of differences in the number of cell deaths per treatment

DISCUSSION:

The combination of doxorubicin with deoxyelephantopin against MCF-7 cells:

Doxorubicin is reported to increase activation of Akt⁹ and NFkB¹⁰ which trigger chemotherapy resistance⁸. Besides doxorubicin also triggers the activity of proteins involved in signal transduction such as PI3K and ERK which also directly triggers the activation of Bcl-XL and Bcl-2 through suppressing Bad activity and overexpression of Pgp which in turn also triggers resistance^21,22. Instead, deoxyelephantopin suppresses activation of NFκβ¹⁸, suppresses PI3K/Akt/mTOR, increases activation of pro-apoptotic proteins (Bad, Bax) and protein caspase (9,8,3), activates the β-Cathenin pathway through suppression of expression c-myc and cyclin D1 thus triggering apoptosis¹³.

The results of a combination cytotoxic test between doxorubicin and deoxyelephantopin showed a higher increase in toxic effects compared to the toxic effects of single doxorubicin. At the smallest concentration of doxorubicin 1/10 IC₅₀ gave viability of 79.77%, but after combined with deoxyelephantopin 1/10 IC₅₀ gave a lower viability of 61.59%. Likewise, the concentration of doxorubicin 1/5 IC₅₀ single treatment gave viability of 70.45%. After combined with deoxyelephantopin 1/10 IC₅₀, viability became 51.36% (Table 1).

The sensitivity of doxorubicin increases with increasing deoxyelephantopin concentrations when combined. The addition of deoxyelephantopin at a concentration of 1/10 IC₅₀, increased the sensitivity of doxorubicin > 4x; the addition of deoxyelephantopin to 1/5 IC₅₀, increases the sensitivity of doxorubicin by almost 7x; the addition of deoxyelephantopin to 1/3 IC₅₀ and 2/5 IC₅₀ increasingly showed a tendency to increase doxorubicin sensitivity (Table 3).

All co-chemotherapy combination formulas (doxorubicin with deoxyelephantopin) showed a synergistic effect with the acquisition value of a combination index (CI) ≤ 0.7. Even the three formulas show a strong synergistic effect with a combination index value ≤ 0.3, namely in the combination of 1/10 IC₅₀ Dox with 1/5 IC₅₀ Deoxy (CI = 0.25); 1/5 IC₅₀ Dox with 1/10 IC₅₀ Deoxy (CI = 0.29); and 1/4 IC₅₀ Dox with 1/10 IC50 Deoxy (CI = 0.28).

Differences in cell death rates for each treatment through "trypan blue" staining:

The synergistic cytotoxic effect of the combination of doxorubicin-deoxyelephantopin is quantitatively confirmed by counting the number of cells that have died. Determination of the number of cells that survive/die in this cytotoxicity test is based on changes in the permeability of the MCF-7 cell membrane through staining with trypan blue. Trypan blue is a dye that is impermeable to cell membranes, an increase in cell membrane permeability resulting in trypan blue can enter the cell. Live cells look intact and clear in color while dead cells are blue due to damage to the cell membrane so the trypan blue dye enters the cell.

The results showed that both doxorubicin and deoxyelephantopin were able to trigger cell death at each IC₅₀ concentration, conversely at each concentration of ½ IC₅₀ doxorubicin and deoxyelephantopin showed lower cell death than controls without treatment (Table 4 and Figure 2). The combination treatment group showed an increasing number of cell deaths. At IC₅₀ concentration concentrations the cell death rate was 2.23x higher than that in the control group, 1.52x higher than the doxorubicin single treatment, and 1.86x higher than the single deoxyelephantopin treatment. Likewise, at the combined concentration of ½ IC₅₀, the cell death rate was 1.7x higher than in controls, 2.12x higher than the single doxorubicin treatment at the IC₅₀ concentration, and 1.82x higher than the single treatment of deoxyelephantopin at the IC₅₀ concentration.

CONCLUSION:

The combination of chemopreventive deoxyelephantopin with doxorubicin chemotherapy is synergistic to increase the sensitivity and effectiveness of doxorubicin chemotherapy against MCF-7 breast cancer cell culture cultures.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 11.05.2020 Modified on 21.06.2020

Research J. Pharm. and Tech. 2021; 14(5):2791-2795.

DOI: 10.52711/0974-360X.2021.00492

1/10 IC₅₀ doxo -1/10 IC₅₀ deoxy	1/5 IC₅₀ doxo - 1/10 IC₅₀ deoxy	1/3 IC₅₀ doxo - 1/10 IC₅₀ deoxy	2/5 IC₅₀ doxo - 1/10 IC₅₀ deoxy
1/10 IC₅₀ doxo - 1/5 IC₅₀ deoxy	1/5 IC₅₀ doxo - 1/5 IC₅₀ deoxy	1/3 IC₅₀ doxo - 1/5 IC₅₀ deoxy	2/5 IC₅₀ doxo - 1/5 IC₅₀ deoxy
1/10 IC₅₀ doxo - 1/3 IC₅₀ deoxy	1/5 IC₅₀ doxo - 1/3 IC₅₀ deoxy	1/3 I_C50 doxo - 1/3 IC₅₀ deoxy	2/5 IC₅₀ doxo - 1/3 IC₅₀ deoxy
1/10 IC₅₀ doxo – 2/5 IC₅₀deoxy	1/5 IC₅₀ doxo – 2/5 IC₅₀ deoxy	1/3 IC₅₀ doxo - 2/5 IC₅₀ deoxy	2/5 IC₅₀ doxo – 2/5 IC₅₀ deoxy