Cell Culture And Chemo Sensitivity Biology Essay

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Epirubincin and Cyclophosphamide are used in this chemo sensitive assay to test the response of the drugs in MCF-7 cell in vitro. Both drugs are anticancer drugs and widely used in the treatment of many cancers such as breast, ovarian, bladder, lungs, thyroid, gastric, soft tissue and oestrogenic sarcomas. In the case study, MCF -7 cells are response to the action of epirubincin but cyclophosphamide doesn't have proper effect on the cell lines in vitro. In practise, the response of cell lines will depend on the correlation between in vitro and vivo. Furthermore, chemo sensitivity of the tumour cells influences the pharmacokinetic action, metabolism of the drugs and drug penetration barrier. Therefore, there is a significant difference in sensitivity for these two drugs when it comes to in vitro assay. Because cyclophosphamide is a pro-drug that needs hepatic microsomal enzyme to become activated. On the other hand, the cytotoxic effect of these two drugs is different in vitro which gives the important clue in the anticancer therapy. The concept emphasizes the importance of the chemo sentitivity assays and it is useful for the future chemotherapeutic regime for research to create new therapy, anti-cancer drugs, their efficacy and therapeutic effects.

Introduction

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The purpose of this practical is to evaluate the response of cancer cells to the two anti-cancer drugs such as Epirubicin and cyclophosphamide by chemo sensitivity assay. Chemo sensitivity assays can also identify new compounds with anti-tumour property and to predict the sensitivity of the tumours for the tumour resistance. Although there are many different types of assay, all have four basic principles. The first step is the isolation of the cells and the second is the incubation of the cells with respective drugs. The third and fourth steps are assessment of cell survival and interpretation of the results respectively.

For the chemo sensitivity assay, cell culture can be done to assess the specific cell line. The aim of cell culture is to manipulate the human genes using human cell lines. Cell culture is the complex process and eukaryotic cells are difficult to culture. The representative cell lines of the cell culture are primary cell line, secondary cell line, immortalized cell line and Hybridization. Cells that are cultured directly from a subject are known as primary cells which undergo one or two division in culture. Secondary cells also from a subject capable of dividing in vitro but their physical characteristic may change after the cells die. Immortalized cells also known as transformed cells and those can continue to grow and divide in vitro when the correct culture are maintained. Nowadays, the Hybridoma technology is used form the hybrid cell line in the cell culture. In this study, the provided cell line is MCF-7 breast cancer cell (Michigan Cancer foundation-7) that is isolated from oestrogen receptor positive mammary adenocarcinoma of the breast cancer cell line.

In this study, we use MTT assay, colorimetric assays for measuring the activity of enzymes. It is a safe, sensitive in vitro assay that allows assessing cell proliferation, apoptosis or necrosis and cell viability reduction. In this assay, tumour cell suspensions are exposed to the tetrazolium chemical compound MTT (3-4-5- dimethylthiazol-2-yl-2-5-diphenyltetrazolium bromide) is added to the wells of the tissue culture plate and incubated. The compound MTT (yellow solution) is reduced by the active cells and become insoluble purple formazan dye crystals. Thus, the sample can be read directly. The optimal wave-length for absorbance is 570 nm but the range between 550 and 600nm may be used. The reduction rate of tetrazolium is directly proportional to the rate of cell proliferation.

Cyclophosphamide is an inactive cyclic phosphamide ester of mechlorethamine and it can transform to an active alkylating metabolites with the help of the hepatic and intracellular enzymes. And then, it adds their alkyl group to the DNA and stops the replication and transcription of DNA. It is mainly activated by the hepatic microsomal enzyme oxidation system (CYP 450) and some peripheral activation. It has some adverse effects on the different systems in which myelosupression is a common haematological side effect and anorexia on the gastrointestinal system. Cyclophosphamide is contraindicated in severe leucopoenia, thrombocytopenia, hepatic or renal dysfunction. It is also contraindicated in pregnancy for the risk of congenital malformations.

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Epirubicin is an anthracycline drug used for chemotherapy. It intercalates into the DNA molecules results in complex formation which inhibits DNA and RNA synthesis. It also inhibits DNA topoisomerase II which is required for DNA function that leads to cell death. Epirubicin also generates free radicals that cause cell and DNA damage. It also have some adverse effects and myelosupression is the most common dose-limiting toxicity and even fatal. Cardio toxicity is the also one of the adverse effects. So, it is contraindicated in severe cardiovascular disease and persistent myelosupression. It is also contraindicated in pregnancy because of the danger of mutagenic and teratogenic effects.

Materials

Materials for Chemo sensitivity assay

96 Well plate

RPMI 1640

MCF - 7

Epirubicin

Cyclophosphamide

MTT assay proliferation kit

DMSO (Dimethyl Sulfoxide)

Universal Microplate Reader (Spectrophotometer)

Filtered Fume Enclosure "Gelman" VERSAFLOW SERIES 2000

Method

MCF-7 breast cancer cell line is supplied by NCI (National Cancer Institute). First, we remove the medium from MCF-7 containing culture flask. And then, 5ml of PBS (phosphate buffered saline) and 1-2ml of trypsin are added to detach the cells from the flask. After a few minutes, the solution is transferred to the falcon tube and centrifuged with 1200 rpm for 1-2 minutes. The supernatant is removed leaving behind the cell pellet. Of which, 180μl of MCF-7 cells are put into the 96 wells micro titre plate and incubated at 37'C with 5% CO2, 95% air and humidity for 24 hours before addition of the experimental drugs. The specific human tumour cell lines to be tested are put in RPMI 1640 medium consisting of 5% foetal bovine serum and 2 mM L-glutamine.

The culture plate containing 96 wells is used to detect the consistency and response of these two drugs. 200 µL of medium (RPMI 1640) is added to the lane 1 of row A to H. To lanes 2 to 12 (A to H) 180 µl if MCF-7 cell suspension is added. 20µl of RPMI 1640 is added to the lane 2 from row A to H to serve as control. 1µM of 20 µl of epirubicin is added to the lane 3 (row A to H). And 20µl (0.5 µM) of epirubicin is added to the lane 4 and the same applies to lane 5 with 0.25 µM of epirubicin. From lane 6 to 12 decreasing concentration of epirubicin like 0.125 µM, 0.0625 µM, and 0.03 µM and so on added respectively. The same procedure is done for cyclophosphamide. Being both of these two drugs are photosensitive, they should be wrapped with aluminium film to prevent light. Moreover the overall procedure should be done in strictly aseptic condition to avoid contamination. The plates are then labelled and incubated for 4 days at 37' C.

Then, 20 µl of MTT is added to each well of the 96 well plates and cultured for 4 hours at 37'C, which is the optimal temperature for MTT to cleave. After 4 hour, 200 µl of supernatant medium plus MTT is carefully removed and discarded from each well because it might disturb the reading. And then, each well is added with 150µl of DMSAO to dissolve the formazan precipitates into the culture medium and mix the solution properly with stir bars. The absorbance reading is taken by EL x 800 Universal micro plate reader at 590nm. The absorbance indirectly indicates the amount of living cells.

Findings and Results

Table 1: Absorbance reading at 490nm, Epirubicin (our group data)

Lane (drug conc)

(μM)

1 (-ve control)

2 (+ve control)

3

1

4

0.5

5

0.25

6

0.125

7

0.0625

8

0.03

9

0.016

10

0.008

11

0.004

12

0.002

A

0.127

0.776

0.287

0.335

0.378

0.386

0.553

0.679

0.699

0.658

0.798

0.887

B

0.129

0.789

0.205

0.287

0.342

0.336

0.438

0.643

0.687

0.687

0.651

0.775

C

0.147

0.761

0.24

0.297

0.324

0.343

0.516

0.493

0.527

0.669

0.674

0.533

D

0.157

0.796

0.242

0.273

0.271

0.404

0.462

0.586

0.603

0.731

0.636

0.785

E

0.131

0.668

0.282

0.273

0.4

0.366

0.485

0.574

0.64

0.72

0.64

0.708

F

0.114

0.743

0.198

0.277

0.356

0.419

0.497

0.593

0.658

0.65

0.612

0.777

G

0.119

0.784

0.216

0.28

0.354

0.412

0.528

0.635

0.656

0.651

0.85

0.731

H

0.149

0.659

0.243

0.332

0.407

0.399

0.536

0.605

0.777

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0.743

0.814

0.71

Mean

0.134

0.747

0.239

0.294

0.354

0.383

0.502

0.601

0.656

0.689

0.709

0.738

Standard deviation

0.015

0.054

0.033

0.025

0.044

0.031

0.039

0.056

0.073

0.038

0.095

0.101

% of cell survival

0%

100%

31.99%

39.36%

47.39%

51.27%

67.20%

80.46%

87.82%

92.24%

94.91%

98.80%

Table 2: Absorbance reading at 490nm, Cyclophosphamide (our group data)

Lane (Drug Conc.)

(μM)

1 (-ve control)

2 (+ve control)

3

1

4

0.5

5

0.25

6

0.125

7

0.0625

8

0.03

9

0.016

10

0.008

11

0.004

12

0.002

A

0.120

0.804

0.811

0.713

0.774

0.737

0.754

0.679

0.756

0.723

0.787

0.737

B

0.116

0.770

0.652

0.738

0.696

0.709

0.730

0.849

0.638

0.785

0.751

0.618

C

0.154

0.760

0.652

0.735

0.630

0.708

0.658

0.638

0.794

0.705

0.688

0.693

D

0.156

0.768

0.641

0.706

0.705

0.755

0.714

0.453

0.662

0.608

0.722

0.477

E

0.110

0.807

0.689

0.715

0.671

0.761

0.726

0.613

0.680

0.662

0.689

0.759

F

0.068

0.770

0.678

0.675

0.634

0.811

0.652

0.747

0.711

0.722

0.572

0.634

G

0.140

0.793

0.714

0.661

0.680

0.597

0.632

0.581

0.646

0.689

0.694

0.660

H

0.119

0.858

0.827

0.841

0.656

0.835

0.787

0.805

0.704

0.722

0.691

0.719

Mean

0.123

0.791

0.708

0.723

0.681

0.739

0.707

0.671

0.699

0.702

0.699

0.662

Standard deviation

0.028

0.032

0.073

0.055

0.046

0.073

0.054

0.128

0.054

0.052

0.063

0.090

% 0f cell survival

0%

100%

89.50%

91.40%

86.09%

93.43%

89.38%

84.83%

88.37%

88.75%

88.37%

83.69%

Table 3: Absorbance readings at 490nm, Epirubicin (given data)

Lane (Drug Conc.)

(μM)

1

(-ve control)

2

(+ve control)

3

1

4

0.5

5

0.25

6

0.125

7

0.0625

8

0.03

9

0.016

10

0.008

11

0.004

12

0.002

A

0.084

0.923

O.214

0.256

0.433

0.522

0.536

0.791

0.947

1.103

1.042

1.568

B

0.079

1.127

0.2

0.272

0.339

0.475

0.462

0.634

1.166

1.403

1.331

1.303

C

0.081

1.067

0.154

0.282

0.369

0.793

0.562

0.311

1.143

1.258

1.215

1.377

D

0.083

1.506

0.165

0.287

0.399

0.602

0.681

0.902

1.225

1.06

1.1

1.556

E

0.081

0.996

0.156

0.26

0.409

0.645

0.743

0.825

1.013

1.354

0.965

1.104

F

0.081

1.829

0.143

0.323

0.374

0.763

0.913

0.659

0.941

1.113

1.543

1.278

G

0.086

1.299

0.154

0.294

0.343

0.75

0.88

0.797

1.261

1.835

1.226

1.209

H

0.133

1.618

0.172

0.344

0.519

0.752

0.751

0.836

0.991

1.049

1.068

1.348

Mean

0.089

1.296

0.163

0.290

0.398

0.663

0.691

0.719

1.086

1.272

1.186

1.343

Standard deviation

0.018

0.326

0.019

0.030

0.058

0.121

0.162

0.188

0.128

0.264

0.186

0.159

% of cell survival

0%

100%

12.5%

22.3%

30.7%

51.1%

53.3%

55.4%

83.8%

98.1%

91.5%

103.6%

Graph1: Relationship of Eiprubicin concentration and percentage of cell survival

Table 4: Absorbance readings at 490nm, Cyclophosphamide (given data)

Lane

(Drug Conc.)(μM)

1 (-ve control)

2 (+ve control)

3

1

4

0.5

5

0.25

6

0.125

7

0.0625

8

0.03

9

0.016

10

0.008

11

0.004

12

0.002

A

0.114

0.676

0.529

0.637

0.665

0.653

0.607

0.671

0.615

0.578

0.623

0.701

B

0.091

0.612

0.491

0.468

0.511

0.503

0.487

0.51

0.523

0.55

0.486

0.603

C

0.088

0.516

0.456

0.409

0.484

0.467

0.482

0.479

0.446

0.51

0.428

0.484

D

0.078

0.576

0.477

0.471

0.49

0.497

0.465

0.482

0.45

0.497

0.445

0.559

E

0.115

0.525

0.508

0.454

0.516

0.528

0.528

0.488

0.454

0.531

0.448

0.519

F

0.113

0.577

0.509

0.438

0.509

0.597

0.57

0.507

0.53

0.51

0.499

0.512

G

0.11

0.627

0.6

0.513

0.579

0.609

0.56

0.592

0.597

0.559

0.55

0.615

H

0.089

0.69

0.495

0.078

0.623

0.309

0.704

0.665

0.68

0.656

0.666

0.652

Mean

0.100

0.600

0.508

0.434

0.547

0.520

0.550

0.549

0.537

0.549

0.518

0.581

Standard deviation

0.015

0.064

0.043

0.159

0.067

0.107

0.079

0.082

0.087

0.051

0.088

0.075

% of cells survival

0%

100%

84.67%

72.33%

91.17%

86.67%

91.67%

91.50%

89.50%

91.50%

86.33%

96.83%

Graph 2: Relationship of Cyclophosphamide concentration and percentage of cell survival

Table 5: IC 50 (inhibitory concentration) values for Epirubicin and cyclophosphamide

Drug

IC 50

Epirubicin

0.125 µM

Cyclophosphamide

0

Discussion

From the above tables and graphs, we can see clearly the response of MCF-7 cell line on these two drugs, epirubicin and cyclophosphamide. In graph 1, epirubicin kills the significant amount of the cells at the concentration of 1 µM in which the percentage of cell survival is about 12.58 %. The percentage of cell survival is directly proportionate to the concentration of epirubicin. This is obliviously seen from the lane 4 to lane 12 in which the percentage of the cell survival rate is dramatically increased because of the cytotoxicity of epirubicin. According to the data in graph 1, we can calculate the IC50 (half maximum inhibitory concentration) which measures the effectiveness of a drug in inhibiting a biological or biochemical function. IC50 for epirubicin is about 0.125 µM. Epirubicin is a member of anthracycline family and causes DNA damage by intercalating of anthracycline portion to the base pairing. It also inhibits the DNA topoisomerase II required for DNA synthesis. It is widely distributed and even can cross the placenta. It is mainly metabolised in liver and excreted via the liver, biliary system and faeces. According to the data from this assay, we can assume that the cytotoxic effect of epirubicin is active in vitro.

When it comes to graph 2 with cyclophosphamide, there is no significant change in the percentage of cell survival apart from the minor differences. Cyclophosphamide is an inactive cyclic phosphamide ester of mechlorethamine and it can only transform with the help of intracellular enzymes to active alkylating agent 4-hydroxycyclophophosphamide, aldophosphamide, and acrolein and phosphoramide mustard. It prevents the cell division primarily by cross-linking DNA and RNA strands. The drug is distributed in most tissues and can cross blood brain barrier and placenta. Metabolism is mainly activated by hepatic microsomal enzyme oxidation system (CYP 450) and active metabolite is phosphoramide mustard/acrolein/4-hydroxy cyclophosphamide. Therefore, we can assume that cyclophosphamide has little or no cytotoxic effect in vitro because the lack of the hepatic microsomal enzyme. Excretion is mainly by kidney and tubular reabsorption occurs.

There are many ways of analysing the effects of drugs on cellular metabolism and morphology; we use MTT chemo sensitivity assays in this study because it is safe and sensitive in vitro. And also, it can measure cell proliferation, apoptosis and cell survivals. Chemo sensitivity assays are intended to predict the sensitivity of various tumour cells to chemotherapeutic agents and identifying the effectiveness of the treatment. In our assay, MCF-7 cells are cultured in plates and the tetrazolium compound MTT is added to the wells. MTT is reduced by metabolically active cells to insoluble purple formazan dye crystals. The rate of tetrazolum reduction is proportional to the rate of cell proliferation. The whole process of these assays should be done under strict aseptic conditions. MTT should be placed in dark. If not, there will be error and difficult for the absorbance reading and the data interpretation.

In these days, the national cancer institute (NCI) introduce the compared method by using the NCI compound's accession number (NSC number) for screening the anti cancer effects of drugs in vitro. The screening involves in two processes, starting with the evaluation of all compounds against 60 cell lines at a single dose of 10 µM. Then the output is analysed by the COMPARE program. Moreover, identification of potential anticancer agents in vitro is required to enhance with demonstration of in vivo animal models to screen for preclinical development.

Conclusion

To conclude that the sensitivity of the two anticancer drugs epirubicin and cyclophosphamide are assessed with MCF-7 cell line by using MTT in vitro chemo sensitivity assay. Although these two drugs play an important role in anticancer regime, their cytotoxic effects are different when it comes to in vitro assay. Epirubicin has cytotoxic effects on MCF-7 cell line in vitro whereas cyclophosphamide does not have because cyclophosphamide needs hepatic microsomal enzyme to become activated. The study suggests that even though these two drugs are well know anticancer drugs but their cytotoxic effects differ significantly in vitro.