{"id":58342,"date":"2024-06-25T10:06:52","date_gmt":"2024-06-25T10:06:52","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=58342"},"modified":"2024-07-03T18:20:05","modified_gmt":"2024-07-03T18:20:05","slug":"comparison-of-anticancer-activity-between-thymoquinone-and-tamoxifen-thymoquinone-tamoxifen-on-mcf-7-cell-line-of-human-breast-cancer-an-invitro-study","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/comparison-of-anticancer-activity-between-thymoquinone-and-tamoxifen-thymoquinone-tamoxifen-on-mcf-7-cell-line-of-human-breast-cancer-an-invitro-study\/","title":{"rendered":"Comparison Of Anticancer Activity Between Thymoquinone And Tamoxifen , Thymoquinone + Tamoxifen On Mcf -7 Cell Line Of Human Breast Cancer \u2013An Invitro Study."},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Breast cancer among women varies hugely in\nincidence around the world.1<\/sup> It is the 2nd<\/sup> most frequent\ncause of death among carcinoma deaths in females which occurs due to metastasis.2,3\n<\/sup>Though there is major progress in the treatment strategies, Breast\ncarcinomas have a high mortality rate.4,5<\/sup><\/p>\n\n\n\n

Cell\nlines play\na major role in studying specific alterations in cell structure.6<\/sup> <\/p>\n\n\n\n

Mechanisms behind drug resistance, which is\nthe main cause for chemotherapy failure in patients with breast cancer,\ncan be studied using the MCF-7 cell line.7,8<\/sup><\/p>\n\n\n\n

Herbert D. Soule developed the MCF-7 cell\nline from a chest wall nodule excision of the patient who was diagnosed to have\nmetastatic disease at Michigan Cancer Foundation.9<\/sup><\/p>\n\n\n\n

Thymoquinone has various properties like\nanti-diabetic, anti-oxidant, anti-allergic, as well as anti-tumor substance\nproperties.10<\/sup> It was established that Thymoquinone promotes\napoptosis in carcinoma cells by XIAP (X-Linked Inhibitor of Apoptosis Protein)\nmediated PKB (Protein Kinase B).11<\/sup> <\/p>\n\n\n\n

Tamoxifen is an estrogen receptor modulator\nthat is non-steroidal. It inhibits the actions of estrogen on breast cancer\ncells by competitively inhibiting estrogen binding to estrogen receptors, which\nis frequently utilized in hormone treatment of receptor-positive breast tumors\nin pre-menopausal as well as post-menopausal females.12<\/sup><\/p>\n\n\n\n

Recent studies have revealed that Tamoxifen\npromotes both cell death as well as cell cycle arrest in carcinoma cells of the\nbreast by activation of ERK1\/2 through signaling induction that causes MAPK and\ncaspase pathway modification in an ER (\u201cEstrogen Receptor\u201d)-positive breast\ncarcinoma cell line (MCF-7) promptly.13,14<\/sup> Tamoxifen is prescribed\nat the dosage of 20mg\/kg body weight per day for the treatment of ER-positive\nbreast cancer.<\/p>\n\n\n\n

Combination therapy plays a major role in\nimproving the efficacy in the treatment of patients having breast carcinoma.15<\/sup>\n<\/p>\n\n\n\n

Hence, in\nthis research, we have assessed the anti-cancerous impact of Tamoxifen to\ninhibit cell proliferation in breast carcinomas, in combination with\nThymoquinone.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

NCCS\n(National Center for Cell\nScience) in Pune provided the MCF-7 cell line. The cells were kept at 37\u00b0C in a\nhumidified medium of 50\u00b5g\/ml CO2<\/sub> in \u201cMinimal Essential Medium\u201d added\nwith 10 percent FBS, streptomycin (100\u00b5g\/ml), and penicillin (100U\/ml).<\/p>\n\n\n\n

In\n24-well plates, cells (1\u00d7105<\/sup>\/well) were plated and incubated at 370<\/sup>C\nwith 5 percent CO2<\/sub> conditions. Samples were introduced at various\nconcentrations and incubated for 24hrs after reaching cell confluence. It was\ntaken from the well after incubation and rinsed in DMEM without serum or\nphosphate-buffered saline (pH 7.4). 0.5percent \u201c3-(4,\n5-dimethyl-2-thiazolyl)-2, 5-diphenyl–tetrazolium bromide (MTT)\u201d was applied\nto 100\u00b5l\/well (5mg\/ml) and incubated for 4hrs. DMSO was applied to all of the\nwells which were used as a blank and the absorbance was recorded at 570nm in\nUV-Spectrophotometer after the incubation period. Measurements were conducted\nand the content needed for a 50percent inhibition (IC50) was calculated and\nevaluated graphically. <\/p>\n\n\n\n

The\nfollowing formula was used to calculate the % cell viability:<\/p>\n\n\n\n

\u201c%Cell viability=A570 of treated cells\/A570\nof control cells\u00d7100\u201d<\/strong><\/p>\n\n\n\n

Cell viability assessments were compared\nbetween the control and test samples in each assay and were plotted on the\ngraphs. <\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

The\nanti-cancerous activity of Thymoquinone, Tamoxifen, and in combination was\nstudied on the MCF-7 cell line at various concentrations that range from\n7.8-1000\u00b5g\/ml and the cell\nviability percentage was measured correspondingly and was tabulated in the table-1.<\/p>\n\n\n\n

The\nfindings revealed the cytotoxic effects of Thymoquinone and Tamoxifen were seen\neven at a minimal concentration on MCF-7 cells using the MTT test.<\/p>\n\n\n\n

The\nmaximum cell viability percentage of Thymoquinone treated cells was found to\nbe   63.75 % at 7.8 \u00b5g\/ml with minimal cell inhibition of\n36.25% and the minimum cell viability percentage of Thymoquinone was 17.66 % at\n1000 \u00b5g\/ml with maximum cell\ninhibition of 82.34% respectively. Similarly, in cell lines treated with\nTamoxifen, the maximum cell viability percentage was 66.54% at 7.8 \u00b5g\/ml\nwith minimal cell inhibition of 33.46 % and the minimum cell viability\npercentage of Tamoxifen was 28.57 % at 1000 \u00b5g\/ml with maximum cell inhibition of 71.43 % respectively. When\ncomparing the MCF-7 cells which were treated with the combination of\nThymoquinone and Tamoxifen it was found that the maximum cell viability\npercentage was 51.95% at 7.8 \u00b5g\/ml with minimal cell inhibition of\n48.05 % and the minimum cell viability percentage of Thymoquinone and Tamoxifen\nwas 16.84% at 1000 \u00b5g\/ml with\nmaximum cell inhibition of 83.16 % respectively.<\/p>\n\n\n\n

The\ncell viability percentage of individual treatment of Thymoquinone, Tamoxifen,\nand the combination (Thymoquinone + Tamoxifen) treatment at various\nconcentrations were plotted and were represented in graph-1. The cell viability\npicture showing the anti-cancerous activity of both individual and combination\ntherapy at various concentrations has been depicted in figure-1.<\/p>\n\n\n\n

Tamoxifen causes a\nchange in plasma membrane permeability due to second messenger formation\nthrough the phospholipase pathway and the sustained activation of protein\nkinase C. This change in cell membrane permeability is responsible for its\napoptotic response.16,17  <\/sup> The increase in Tamoxifen concentration\nincreases the intensity of MCF7 mitochondrial membrane permeability, which leads\nto the loss of mitochondrial membrane potential and the release of cytochrome C\nfrom the mitochondria causing cytotoxic effects in MCF-7 breast cancer cell\nlines.18   <\/sup>Studies have suggested that tamoxifen\nis efficacious with good tolerance and bioavailability.19-21<\/sup><\/p>\n\n\n\n

Thymoquinone\ncauses apoptosis in cancer cells by various multiple target modulation such as\np53-dependent, and p53-independent pathway, activation of caspases, increases\nin p53 expression, anti-apoptotic Bcl-2 up-regulation and decrease in cyclins\nB1 and D1.22 <\/sup>Recent studies have revealed that NF-kB is a potential\ntarget for inducing apoptosis in cancer cells.23<\/sup><\/p>\n\n\n\n

Metastasis is the major limitation of\nTamoxifen treatment in breast carcinoma patients which occurs due to\noverexpression of Transforming Growth Factor-\u03b2 (TGF-\u03b2) on prolonged therapy.24-26<\/sup><\/p>\n\n\n\n

Moreover, resistance to Tamoxifen treatment\nis another consequence of reducing its efficacy. Inquisitively, shreds of\nevidence revealed that in chronic treatments -Tamoxifen acts as an\nestrogen-like structure, promoting tumor progression.27<\/sup><\/p>\n\n\n\n

Tamoxifen concentration is reduced in breast\ncarcinoma cells owing to the presence of P-glycoprotein, causing a treatment\ncompromise. This was believed to be a cause for the expansion of innate\nresistance in breast carcinoma patients treated with Tamoxifen.11<\/sup><\/p>\n\n\n\n

Considering the above-mentioned concepts, this study\nspecifies the need for combination therapy in place of monotherapy. It has\nrecently been proved that Thymoquinone produces a synergistic impact on\nTamoxifen through XIAP- mediated Akt regulation in estrogen receptor-positive\nand negative breast carcinomas when compared to monotherapy with Tamoxifen. In addition to this, decrease in the levels of\nanti-apoptotic Bcl-2 and Bcl-xL expression, and an increase in the levels of\npro-apoptotic Bax, p27, cytosolic AIF and cytochrome C proteins is responsible\nfor increased apoptotic effect in MCF-7 treated with Thymoquinone and Tamoxifen. Besides they induce apoptosis by activation\nof Caspase-9 and by decreased level of p-Bad (BCL2 Antagonist of cell Death)<\/em>, p-MAPK and p-GSK-3\u03b2\nin Akt pathway.11<\/sup><\/p>\n\n\n\n

Literature states that though Thymoquinone\nhas anti-cancerous activity, evidence revealed that the addition of this drug\nwith Tamoxifen improved the efficacy.11<\/sup> <\/p>\n\n\n\n

In this study, it was observed that combination therapy of Thymoquinone and Tamoxifen had better efficacy and additive anti-cancerous activity in the MCF-7 cell line than sole therapy.<\/p>\n\n\n\n

Table 1: Anticancer effect of Thymoquinone, Tamoxifen and Thymoquinone + Tamoxifen on<\/strong> MCF 7 cell line<\/strong> <\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

S.No<\/strong><\/p>\n<\/td>\n

\n

Concentration
(\u00b5g\/ml)<\/strong><\/p>\n<\/td>\n

\n

Dilutions<\/strong><\/p>\n<\/td>\n

\n

Thymoquinone<\/strong><\/p>\n<\/td>\n

\n

Tamoxifen<\/strong><\/p>\n<\/td>\n

\n

Thymoquinone
+ Tamoxifen<\/strong><\/p>\n<\/td>\n<\/tr>\n

 <\/td>\n\n

 <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Absorbance
(O.D*)<\/strong><\/p>\n<\/td>\n

\n

Cell
Viability
(%)<\/strong><\/p>\n<\/td>\n

\n

Absorbance
(O.D*)<\/strong><\/p>\n<\/td>\n

\n

Cell
Viability
(%)<\/strong><\/p>\n<\/td>\n

\n

Absorbance
(O.D*)<\/strong><\/p>\n<\/td>\n

\n

Cell
Viability
(%)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

1000<\/p>\n<\/td>\n

\n

Undiluted<\/p>\n<\/td>\n

\n

0.235<\/p>\n<\/td>\n

\n

17.66<\/p>\n<\/td>\n

\n

0.380<\/p>\n<\/td>\n

\n

28.57<\/p>\n<\/td>\n

\n

0.224<\/p>\n<\/td>\n

\n

16.84<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

500<\/p>\n<\/td>\n

\n

1:1<\/p>\n<\/td>\n

\n

0.332<\/p>\n<\/td>\n

\n

24.96<\/p>\n<\/td>\n

\n

0.435<\/p>\n<\/td>\n

\n

34.21<\/p>\n<\/td>\n

\n

0.290<\/p>\n<\/td>\n

\n

21.80<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

250<\/p>\n<\/td>\n

\n

1:2<\/p>\n<\/td>\n

\n

0.413<\/p>\n<\/td>\n

\n

31.05<\/p>\n<\/td>\n

\n

0.528<\/p>\n<\/td>\n

\n

39.69<\/p>\n<\/td>\n

\n

0.357<\/p>\n<\/td>\n

\n

26.84<\/p>\n<\/td>\n<\/tr>\n

\n

 4<\/p>\n<\/td>\n

\n

125<\/p>\n<\/td>\n

\n

1:4<\/p>\n<\/td>\n

\n

0.496<\/p>\n<\/td>\n

\n

37.29<\/p>\n<\/td>\n

\n

0.602<\/p>\n<\/td>\n

\n

45.26<\/p>\n<\/td>\n

\n

0.424<\/p>\n<\/td>\n

\n

31.87<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

62.5<\/p>\n<\/td>\n

\n

1:8<\/p>\n<\/td>\n

\n

0.579<\/p>\n<\/td>\n

\n

43.53<\/p>\n<\/td>\n

\n

0.673<\/p>\n<\/td>\n

\n

50.60<\/p>\n<\/td>\n

\n

0.490<\/p>\n<\/td>\n

\n

36.84<\/p>\n<\/td>\n<\/tr>\n

\n

6<\/p>\n<\/td>\n

\n

31.2<\/p>\n<\/td>\n

\n

1:16<\/p>\n<\/td>\n

\n

0.665<\/p>\n<\/td>\n

\n

50.00<\/p>\n<\/td>\n

\n

0.741<\/p>\n<\/td>\n

\n

55.71<\/p>\n<\/td>\n

\n

0.557<\/p>\n<\/td>\n

\n

41.87<\/p>\n<\/td>\n<\/tr>\n

\n

7<\/p>\n<\/td>\n

\n

15.6<\/p>\n<\/td>\n

\n

1:32<\/p>\n<\/td>\n

\n

0.748<\/p>\n<\/td>\n

\n

56.24<\/p>\n<\/td>\n

\n

0.814<\/p>\n<\/td>\n

\n

61.20<\/p>\n<\/td>\n

\n

0.624<\/p>\n<\/td>\n

\n

46.91<\/p>\n<\/td>\n<\/tr>\n

\n

8<\/p>\n<\/td>\n

\n

7.8<\/p>\n<\/td>\n

\n

1:64<\/p>\n<\/td>\n

\n

0.848<\/p>\n<\/td>\n

\n

63.75<\/p>\n<\/td>\n

\n

0.885<\/p>\n<\/td>\n

\n

66.54<\/p>\n<\/td>\n

\n

0.691<\/p>\n<\/td>\n

\n

51.95<\/p>\n<\/td>\n<\/tr>\n

\n

9<\/p>\n<\/td>\n

\n

Cell control<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

1.330<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n

\n

1.330<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n

\n

1.330<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*O.D- Optical Density<\/em><\/p>\n\n\n\n
\"\"<\/td>\n\n

Graph 1: Graphical representation of cell viability percentage of individual treatment of Thymoquinone, Tamoxifen, and the combination (Thymoquinone + Tamoxifen) treatment at various concentrations.<\/strong><\/p>\n

<\/p>\n

Click here to view Graph<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Anticancer effect of Thymoquinone on MCF 7 cell line<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In this\nstudy, it is determined that the combined effect of Thymoquinone and Tamoxifen\nhas the better activity of cell inhibitory on MCF 7 cell line growth when\ncompared with the individual effect of Thymoquinone and Tamoxifen. Hence the\nabove combination can be considered for better efficacy in the treatment of\nbreast carcinoma patients. Further In-Vivo evaluations are needed in the future\nto aid its anti-cancerous effect. <\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

None to declare<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

There is no any\nconflict of interest between authors.<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

There is no funding Sources<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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