Reducing The Side Effects Of Colchicine Biology Essay

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Cancer is basically a disorder charecterised by uncontrolled division of cells of any particular tissue or organ. This division of cells occurs by mitosis and the drugs which can block this mitotic division of cancer cells are being used from a long period of time in treatment of cancer, particularly those drugs which target microtubules were found to be the most effective. But these drugs cannot cure cancer completely because of development of resistance to these drugs by cancer cells after continuos usage of these drugs for a long time. But colchicine is one such drug which irrevesibly binds to microtubules, thus can overcome the problem of drug resistance. But colchicine is not used in cancer treatment because of its severe side effects which some times can even lead to death. These side effects are mainly due to the lack of specificity of action by colchicine which causes mitotic arrest even in normal body cells along with cancer cells. We can overcome this problem by reducing the concentration of colchicine to such an extent at which at which it acts mainly on cancer cells with minimal effect on cancer cells, because microtubules are prominent in rapidly dividing cells when compared to slowly dividing normal body cells thus rapidly dividing cells are more suseptable to colchicine. In this study we tried to find out the least possible concentration of colchicine which can effectively block the division of rapidly dividing cells with minimal or no effect on slowly dividing cells.

Keywords: colchicine, microtubules, yeast, colorimeter, incubation

1. Introduction:

Colchicine is a powerful antimitotic agent obtained from the plant colchicum autumnale. It blocks mitosis by irreversibly binding to microtubules(Amos et al., 1974) at specific binding site called as the colchicine site(Joseph Bryan et al., 1972). By binding to microtubles it induces microtubule depolymerization at high concentrations and it suppress microtubule dynamics at low concentrations and because of the fact that spindle fibers(Campbell et al., 2005) are formed by the union of microtubules, colchicine ultimately blocks the spindle fiber formation. As we all know spindle fibers are responsible for the seggregation of chromosomes in mitosis, thus colchicine ultimately brings mitosis to a halt when it is still in metaphase which eventually leads to apoptotic(Jordan et al., 1999) cell death.

Along with the regular antimitotic actions colchicine is also capable of reproducing the cellular changes induced in cells by x rays hence called as radiomimetic drug(British Medical Journal., 1950). Because of its irreversible binding to microtubules it can also overcome the problem of drug resistance which currently is a major limiting factor for most of the microtubule targeting drugs.

Due to all these properties colchicine should prove to be the most effective anti cancer agent. Instead it is not at all used in the treatment of cancer because of its severe side effects which sometimes can even be deadly. These side effects can be heamatological(Rowinsky et al., 1997) and neurological(Lobert et al.,1997) and can range from nausea and vomiting to central neurotoxicities and myelosuppression and can sometimes lead to death. These side effects are mainly because of the action of colchicine on normal cells along with the cancerous cells. The effects like myelosuppression are mainly due to the action of colchicine on rapidly dividing haemopoietic cells.

These side effects can be reduced by reducing the dose of colchicine to least possible dose at which it is effective only on rapidly dividing cells like cancer cells. This happens because colchicine targets microtubules and microtubules are formed only in dividing cells. So, the rapid the cell divides more it is susceptable to colchicine. Generally the rate of division of the cancer cells is very high when compared to any other cells in the body including the cells in the bone marrow, because of which cancer cells are more suseptable to colchicine in low concentrations than normal body cells.

In this study we have studied the effect of colchicine on yeast cells in two different culture mediums of which one was incubated in optimum temperature(26°c) conditions to simulate rapidly dividing cancer cells and other culture was maintained at sub-optimal temperature(10°c) to simulate normal body cells because yeast grows slowly at reduced temperatures.

Reasons for choosing yeast as a model organism:

Yeast is a eukaryotic organism and the cells present in the human body are also eukaryotic in nature.

The cell cycle regulatory mechanisms found in yeast cells are very simillar to that found in human cells.

It is very easy to culture yeast in the laboratory when compared to other microbes.

All the stages of mitosis that are seen in humans can be seen in yeasts.

Because yeast divides very rapidly they can be used to represent cancer cells.

2. Materials and methods:

2.1. yeast cells(Saccharomyces cerevisae): we bought yeast cells(in dehydrated form) from Hainan Zhonghong Yeast Co.,Ltd located at No.2 Shimao East Haikou Hainan China.

2.2. Preperation of culture media(potato dextrose agar and potato dextrose broth(Lawrence et al., 1997):

The dextrose required for preperation of media was bought from S.G.R Engineers located at 69b/1, Ram Nagar 2nd Main Road, Peravallur, Jawahar Nagar Post, Chennai - 600082, Tamil Nadu, India and agar agar required for the preperation of media was bought from Marine chemicals located at Deepa Building, Santo Gopalan Road, Chullikal, Kerala India. The culture medium was prepared by preparing a potato infusion by boiling 300gm of sliced (washed but unpeeled) potatoes in water for 30 minutes and then decanting or straining the broth through cheesecloth then distilled water was added such that the total volume of the suspension is one litre, followed by addition of 20g of dextrose which was followed by addition of 20gm of agar agar(solidifying agent) for the preperation of culture plates and agar agar was excluded in the preperation of liquid broth and then the medium was sterilized by autoclaving at 15psi for 15 minutes and the medium containing agar was poured into Petri-plates when hot and was allowed to solidify to form culture plates to be used in plate count technique and liquid broth was poured in culture tubes.

2.3. Preperation of colchicine dilutions:

We bought colchicine from pharmanza india private limited situated at 70/1, Gidc Estate, Kansari, Khambhat - 388630, Gujarat. Then we prepared diluted solutions of colchicine by suspending (it cannot be dissolved because 1gm of colchicine is soluble in 25ml of water) 100gm of colchicine in 100ml of distilled water to obtain a colchicine solution of concentration 1:1, 10ml of this suspension was diluted 10 times to obtain a concentration of 1:10, in this way we continued the dilution serially and prepared solutions of dilution upto 1:1010 .

2.4. Inoculation of culture media with yeast cells:

The yeast cells obtained in dehydrated form were hydrated by adding water and were allowed to grow in a broth culture. From this broth culture a single yeast cells were isolated using a micro pippete and were inoculated in all the broth culture tubes meant for study. These cultures were then incubated for the growth of yeast cells.

2.5. Turbidimetric estimation of cell numbers:

For the estimation of turbidity we bought a colorimeter from Chennai laboratories situated at 4a, Gowri Chitra Garden, Arcat Road, Near Vadapalani Bus Dipo, Chennai, Tamil Nadu. using this instrument we first measured the turbidity of potato dextrose broth which was not inoculated with yeast cells in terms of optical density at a wavelength of 420nm, then we stirred the broth cultures well for uniform distribution of cells throughout the culture tube and then we have withdrawn a 2ml of the broth and diluted it to about 10 times its volume with distilled water and measured the turbidity in terms of optical density. As the number of cells in the liquid broth increased the optical density of the liquid broth also increased. It is very important to dilute the liquid broth before turbidimetry because the liquid broth is very turbid if used as it is and because of this, the sensitivity of the instrument was reduced and it could not detect a small change in the cell numbers.

2.6. Estimation of cell numbers by plate count method(M W LeChevallier et al., 1980):

In this method a small sample of 2ml was withdrawn from 20ml of broth culture containing the yeast cells and it was diluted in the range of 1:10 to 1:1,00,000 by serial dilution method and 1ml of this diluted solution was spread on the culture plate(known as spread plate technique) prepared in the earlier step and was incubated for 12 hours and during this period each cell developed into a colony and the number of colonies formed were counted ,thus we obtained the number of cells present in 1ml of diluted solution which we multiplied with total volume of diluted solution and we then multiplied this value with the dilution factor and obtained the total number of cells present in undiluted potato dextrose broth culture. But generally a single cell may not be spred over the culture plate, thus we cannot say that the colony has formed from a single cell. Hence these are called as colony forming units(CFU'S) instead of cells.

2.7. Treatment of broth culture with colchicine:

The diluted solutions of colchicine prepared earlier were used for this purpose and first of all a dilution of 1:1 was added in six different doses of 1ml, 2ml, 4ml, 6ml, 8ml, 10ml and each dose was added to a separate culture tube in set A and mixed well, thus keeping the dilution constant but changing the dose. In the same manner other culture tubes were also treated with remaining dilutions of colchicine. As mentioned earlier a total of 10 different dilutions were prepared and each dilution was adminstered in six different doses thus the effect of colchicine was studied on sixty six different culture tubes in set A which were incubated at 10°c.

Same procedure was followed for another sixty six culture tubes in set B which were incubated at 26°c.

3. Results and discusssions:

3.1. Plotting of caliberation curve:

Initially potato dextrose broth was taken in 12 culture tubes numbered from 1-12 and each one of the 12 culture tubes were inoculated with a single Saccharomyces cerevisae cell isolated under a high power microscope using a micro pippete and were incubated at 26°c and each test tube was incubated for different time periods ranging from one hour to 12 hours and number of cells present in the each culture tube were counted by plate count method using spread plate technique and the same test tubes were subject to turbidimetric analysis(for this the broth containing yeast cells was diluted to 10 times the original value to get proper readings in the colorimeter because the potato dextrose broth as such is very turbid and if we use it directly the sensitivity of the instrument is lost) using a colorimeter at a wavelength of 420nm. The cell count varied according to the duration of incubation and these observations are tabulated in table-1 and then the turbidimetric values were plotted against the number of cells and a caliberation curve was obtained by which we could turbidimetrically estimate the number of cells in the broth culture.

Table-1: Duration of incubation of each culture tube and the number of cells in each culture tube found by plate count method and the corresponding optical density values found with the help of colorimeter

S.No

Duration of incubation

Number of cells(CFU'S) counted by

plate count method

Optical density of liquid broth after diluting it ten times its original volume

1

Culture tube not inoculated with yeast cell

_

0.09

2

1 hour

Could not be estimated even without dilution because of very less cells

0.09

3

2 hours

Could not be estimated even without dilution because of very less cells

0.09

4

3 hours

Could not be estimated even without dilution because of very less cells

0.10

5

4 hours

Number of cells is found to be 300 counted at 1:10 dilution

0.13

6

5 hours

Number of cells is found to be 1,000 at 1:10 dilution

0.19

7

6 hours

Number of cells is found to be 4,000 counted at 1:100 dilution

0.25

8

7 hours

Number of cells is found to be 16,000 counted at 1:100 dilution

0.42

9

8 hours

Number of cells is found to be 104-6 counted at 1:1000dilution

0.63

10

9 hours

Number of cells is found to be 105-2 counted at 1:10,000 dilution

0.85

11

10 hours

Number of cells is found to be 106 counted at 1:10,000dilution

1.21

12

11 hours

Number of cells is found to be 106-4 counted at 1:100,000 dilution

1.82

13

12 hours

Number of cells is found to be 107 counted at 1:100,000 dilution

2.31

Due to the broad range of data the caliberation curve is split into three parts and is represented as three different curves

Fig-1: Caliberation curve with optical density found using colorimeter plotted against number of cells found by plate count method used to find cell numbers upto 16,000.

Fig-2: Caliberation curve with optical density found using colorimeter plotted against number of cells found by plate count method used to find cell numbers in the range of 16,000 - 10,00,000.

Fig-3: Caliberation curve with optical density found using colorimeter plotted against number of cells found by plate count method used to find cell numbers in the range of 10,00,000 - 1,00,00,000.

3.2. Estimation of effect of temperature on growth of yeast cells:

Saccharomyces cerevisae was cultured on potato dextrose broth taken in two different culture tubes of 20ml capacity. One culture tube was incubated at a temperature of 26°c and other was incubated at temperature of 10°c for 12 hours. The number of cells in each culture were estimated turbidimetrically after 12 hours and the number of cells in the tube incubated at 10°c was found to be around 104 and the number of cells in the culture tube which was incubated at 26°c was found to be around 107 thus at reduced temperature the growth rate of yeast cells is found to be less.

3.3. Effect of various concentrations of colchicine on slowly and rapidly dividing cells:

The yeast cells which are incubated at 10°c divide slowly and represent normal body cells and the yeast cells which are incubated at 26°c divide at a much faster rate and represent cancer cells.

Potato dextrose broth was taken in 132 culture tubes of 20ml capacity and the culture tubes were numbered from 1-132 and all culture tubes were inoculated with Saccharomyces cerevisae and the culture tubes which are numbered 1-66 were incubated at 10°c and are considered as set A and the culture tubes which are numbered from 66-132 were incubated at 26°c are considered as set B. Both the sets of test tubes were incubated for ten hours and the number of cells in the broth culture were estimated turbidimetrically with the help of the caliberation curve and then all of these culture tubes were treated with six different doses of 1ml, 2ml, 4ml, 6ml, 8ml, 10ml of each dilution of colchicine ranging from 1:1 to 1:1010 and were again incubated for another two hours and the number of cells in the culture were estimated to study the extent of mitotic arrest caused by different concentrations of colchicine on rapidly dividing cells(set B) and slowly dividing cells(set A). The results of this test are tabulated in table-2.

Table-2: Effect of different dilutions of colchicine on division of yeast cells maintained at 10°c and 26°c

S.No

Set A or B

Dilution of colchicine used

Dose in ml

Optical density

Number of cells before treatment with colchicine

Optical density

Number of cells after treatment with colchicine

1

Set A

1:1

1

0.3

6,000

0.3

6,000

2

Set A

1:1

2

0.3

6,000

0.3

6,000

3

Set A

1:1

4

0.3

6,000

0.3

6,000

4

Set A

1:1

6

0.3

6,000

0.3

6,000

5

Set A

1:1

8

0.3

6,000

0.3

6,000

6

Set A

1:1

10

0.3

6,000

0.3

6,000

7

Set A

1:10

1

0.3

6,000

0.3

6,000

8

Set A

1:10

2

0.3

6,000

0.3

6,000

9

Set A

1:10

4

0.3

6,000

0.3

6,000

10

Set A

1:10

6

0.3

6,000

0.3

6,000

11

Set A

1:10

8

0.3

6,000

0.3

6,000

12

Set A

1:10

10

0.3

6,000

0.3

6,000

13

Set A

1:100

1

0.3

6,000

0.3

6,000

14

Set A

1:100

2

0.3

6,000

0.3

6,000

15

Set A

1:100

4

0.3

6,000

0.3

6,000

16

Set A

1:100

6

0.3

6,000

0.3

6,000

17

Set A

1:100

8

0.3

6,000

0.3

6,000

18

Set A

1:100

10

0.3

6,000

0.3

6,000

19

Set A

1:1000

1

0.3

6,000

0.3

6,000

20

Set A

1:1000

2

0.3

6,000

0.3

6,000

21

Set A

1:1000

4

0.3

6,000

0.3

6,000

22

Set A

1:1000

6

0.3

6,000

0.3

6,000

23

Set A

1:1000

8

0.3

6,000

0.3

6,000

24

Set A

1:1000

10

0.3

6,000

0.3

6,000

25

Set A

1:104

1

0.3

6,000

0.3

6,000

26

Set A

1:104

2

0.3

6,000

0.3

6,000

27

Set A

1:104

4

0.3

6,000

6,000

28

Set A

1:104

6

0.3

6,000

0.3

6,000

29

Set A

1:104

8

0.3

6,000

0.3

6,000

30

Set A

1:104

10

0.3

6,000

0.3

6,000

31

Set A

1:105

1

0.3

6,000

0.31

6,000-7000

32

Set A

1:105

2

0.3

6,000

0.31

6,000-7000

33

Set A

1:105

4

0.3

6,000

0.31

6,000-7000

34

Set A

1:105

6

0.3

6,000

0.3

6,000

35

Set A

1:105

8

0.3

6,000

0.3

6,000

36

Set A

1:105

10

0.3

6,000

0.3

6,000

37

Set A

1:106

1

0.3

6,000

0.35

8,000

38

Set A

1:106

2

0.3

6,000

0.35

8,000

39

Set A

1:106

4

0.3

6,000

0.31

6000-7000

40

Set A

1:106

6

0.3

6,000

0.31

6000-7000

41

Set A

1:106

8

0.3

6,000

0.3

6,000

42

Set A

1:106

10

0.3

6,000

0.3

6,000

43

Set A

1:107

1

0.3

6,000

0.34

8,000

44

Set A

1:107

2

0.3

6,000

0.34

8,000

45

Set A

1:107

4

0.3

6,000

0.34

8,000

46

Set A

1:107

6

0.3

6,000

0.33

7,000-8,000

47

Set A

1:107

8

0.3

6,000

0.33

7000-8,000

48

Set A

1:107

10

0.3

6,000

0.33

7000-8,000

49

Set A

1:108

1

0.3

6,000

0.35

8,000-9,000

50

Set A

1:108

2

0.3

6,000

0.35

8,000-9,000

51

Set A

1:108

4

0.3

6,000

0.35

8,000-9,000

52

Set A

1:108

6

0.3

6,000

0.35

8,000-9,000

53

Set A

1:108

8

0.3

6,000

0.34

8,000

54

Set A

1:108

10

0.3

6,000

0.34

8,000

55

Set A

1:109

1

0.3

6,000

0.35

8,000-9,000

56

Set A

1:109

2

0.3

6,000

0.35

8,000-9,000

57

Set A

1:109

4

0.3

6,000

0.35

8,000-9,000

58

Set A

1:109

6

0.3

6,000

0.35

8,000-9,000

59

Set A

1:109

8

0.3

6,000

0.35

8,000-9,000

60

Set A

1:109

10

0.3

6,000

0.35

8,000-9,000

61

Set A

1:1010

1

0.3

6,000

0.37

9,000-10,000

62

Set A

1:1010

2

0.3

6,000

0.37

9,000-10,000

63

Set A

1:1010

4

0.3

6,000

0.37

9,000-10,000

64

Set A

1:1010

6

0.3

6,000

0.37

9,000-10,000

65

Set A

1:1010

8

0.3

6,000

0.37

9,000-10,000

66

Set A

1:1010

10

0.3

6,000

0.37

9,000-10,000

67

Set B

1:1

1

1.0

106

1.0

106

68

Set B

1:1

2

1.0

106

1.0

106

69

Set B

1:1

4

1.0

106

1.0

106

70

Set B

1:1

6

1.0

106

1.0

106

71

Set B

1:1

8

1.0

106

1.0

106

72

Set B

1:1

10

1.0

106

1.0

106

73

Set B

1:10

1

1.0

106

1.0

106

74

Set B

1:10

2

1.0

106

1.0

106

75

Set B

1:10

4

1.0

106

1.0

106

78

Set B

1:10

6

1.0

106

1.0

106

79

Set B

1:10

8

1.0

106

1.0

106

80

Set B

1:10

10

1.0

106

1.0

106

80

Set B

1:100

1

1.0

106

1.0

106

81

Set B

1:100

2

1.0

106

1.0

106

82

Set B

1:100

4

1.0

106

1.0

106

83

Set B

1:100

6

1.0

106

1.0

106

84

Set B

1:100

8

1.0

106

1.0

106

85

Set B

1:100

10

1.0

106

1.0

106

86

Set B

1:1000

1

1.0

106

1.0

106

87

Set B

1:1000

2

1.0

106

1.0

106

88

Set B

1:1000

4

1.0

106

1.0

106

89

Set B

1:1000

6

1.0

106

1.0

106

90

Set B

1:1000

8

1.0

106

1.0

106

91

Set B

1:1000

10

1.0

106

1.0

106

91

Set B

1:104

1

1.0

106

1.0

106

92

Set B

1:104

2

1.0

106

1.0

106

93

Set B

1:104

4

1.0

106

1.0

106

94

Set B

1:104

6

1.0

106

1.0

106

95

Set B

1:104

8

1.0

106

1.0

106

96

Set B

1:104

10

1.0

106

1.0

106

97

Set B

1:105

1

1.0

106

1.0

106

98

Set B

1:105

2

1.0

106

1.0

106

99

Set B

1:105

4

1.0

106

1.0

106

100

Set B

1:105

6

1.0

106

1.0

106

101

Set B

1:105

8

1.0

106

1.0

106

102

Set B

1:105

10

1.0

106

1.0

106

103

Set B

1:106

1

1.0

106

1.18

106 -2

104

Set B

1:106

2

1.0

106

1.18

106 -2

105

Set B

1:106

4

1.0

106

1.0

106

106

Set B

1:106

6

1.0

106

1.0

106

107

Set B

1:106

8

1.0

106

1.0

106

108

Set B

1:106

10

1.0

106

1.0

106

109

Set B

1:107

1

1.0

106

1.38

106 -3

110

Set B

1:107

2

1.0

106

1.18

106 -2

111

Set B

1:107

4

1.0

106

1.08

106 +8-105

112

Set B

1:107

6

1.0

106

1.0

106

113

Set B

1:107

8

1.0

106

1.0

106

114

Set B

1:107

10

1.0

106

1.0

106

115

Set B

1:108

1

1.0

106

1.72

106 -4

116

Set B

1:108

2

1.0

106

1.38

106 -3

117

Set B

1:108

4

1.0

106

1.38

106 -3

118

Set B

1:108

6

1.0

106

1.18

106 -2

119

Set B

1:108

8

1.0

106

1.08

106 +8-105

120

Set B

1:108

10

1.0

106

1.0

106

121

Set B

1:109

1

1.0

106

1.84

106 -5

122

Set B

1:109

2

1.0

106

1.38

106 -3

123

Set B

1:109

4

1.0

106

1.38

106 -3

124

Set B

1:109

6

1.0

106

1.38

106 -3

125

Set B

1:109

8

1.0

106

1.18

106 -2

126

Set B

1:109

10

1.0

106

1.07

106 +7-105

127

Set B

1:1010

1

1.0

106

2.1

106 -7

128

Set B

1:1010

2

1.0

106

1.92

106 -6

129

Set B

1:1010

4

1.0

106

1.92

106 -6

130

Set B

1:1010

6

1.0

106

1.84

106 -5

131

Set B

1:1010

8

1.0

106

1.72

106 -4

132

Set B

1:1010

10

1.0

106

1.72

106 -4

From the results we can see that as the concentration of colchicine decreases it is more effective on rapidly dividing cells than slowly dividing cells because the microtubules are more prominent in rapidly dividing cells . Hence the mitotic arrest is more in rapidly dividing cells because of which the cell numbers were less in case of rapidly dividing cells at the end of incubation period when compared to slowly dividing cells.

4. Conclusions:

In this experiment we tried to artificially simulate the behaviour of cancer cells and normal boby cells and studied the effect of colchicine on these cells, by adminstering it in different concentrations and tried to find out the least concentration of colchicine at which it can effectively cause mitotic arrest in cancer cells without effecting normal cells. We also tried to determine the effect of doseage on effectiveness of colchicine even in least concentrations.

In this experiment we found that colchicine, if given in high concentration is lethal to both slowly dividing cells and rapidly dividing cells which represent normal body cells and cancer cells respectively. But when the concentration was reduced to as low as 1:1010 , the overall intensity of action was less but even in this case the intensity of action was more on rapidly dividing cells when compared to slowly dividing cells because microtubules are more prominent in rapidly dividing cells when compared to slowly dividing cells, but it is clear from the experiment that even at this concentration there was a considerable impact of dose on the cells.

But the least concentration of colchicine which was found to be effective on yeast cells was at a dilution of 1:109 at a dose of 10ml , because at this concentration the anti mitotic activity was very high on rapidly dividing cells and effect was very less on slowly dividing cells which is clear from the results.

Eventhough these observations cannot be applied directly for dose determination for human beings, these can be used as a base for testing in the animal models. But the main problem with the adminstration in such low doses in animals is that the volume of distribution is very high in animals and because of this only a very low amount of colchicine actually reaches the cancer cells. If we can find ways to overcome this problem, then we will be able to use colchicine for the treatment of cancer.

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