EFFECTS OF KACIMEX ON CANCER IN EXPERIMENT

EFFECTS OF KACIMEX ON EXPERIMENTAL LIVER CANCER IN WHITE Mice

AND THE EFFECTS OF THE DRUG ON THE IMMUNE SYSTEM OF TUM CARRYING Mice

Prof.Huangren Bin.PhD *; Dr Nguyen Phu Kieu PhD ** ;Prof. PhamKim Man,PhD**

*Institute of Clinical Pharmacology - Naning Medical University - China.

** Vietnam Institute for Research and Treatment of Serious Diseases.

I. MATERIALS AND METHODS

1. Materials

1.1. Experimental drugs and chemicals:

- Kacimex, a drug produced by Que Lam Company Limited, Vietnam Institute for Research and Treatment of Serious Diseases.

- Cyclophosphamide, drug produced by Hang Thuy Company, Jiangsu, number 06041521

- 5-fluorouracil

-Dimethylthiazole, product manufactured by Sigma Company, USA

- Environment RPMI-1640 (Roswell Park Memorial Institute), a product produced by Hyclone Company

- Fresh calf serum, produced by Tu Quy Thanh Company, Hang Chau

1.2. Devices:

- Automatic ELISA reader, Microfluid mixing device, Inverted phase contrast microscope.

- CO2 incubator

- Super clean culture chamber

- Deep refrigerator

1.3. Experimental animals

- White mice, average weight 18-22g, half male and female, provided by the experimental animal breeding center, Guangxi University of Medicine. Approval number: Que SCXK-2003.

- Mouse liver cancer cell line H22, 3 human tumor cell lines 7404, SGC-7901, HEPG2 are stored by the Institute of Pharmacy, Guangxi University of Medicine.

2. Method

Use the MTT colorimetric method with Dimethylthiazole to determine the inhibitory effect of Kacimex on cell proliferation.

Use peptidase to treat 3 human tumor cell lines 7404, SGC-7901, HEPG2 while growing at multiplicity, then use RPMI-1640 medium adding 10% fresh fetal bovine serum to prepare a suspension. cells with a concentration of 107 cells/ml, put into a 96-hole plastic culture plate, each hole is 190 µl, incubated in a culture chamber of 5% CO2, 37oC, 95% humidity for 8 hours. Design the template into:

- Comparison group: only add 5-fluorouracil

- Control group: only add RPMI-1640 medium

- Experimental group: differentiate between adding different concentrations of 5-fluorouracil and Kacimex medicine

Culture for 48 hours, add 10µl Thiazole green (5g/L), continue culturing for another 4 hours under the same conditions above, quickly turn the plastic culture plate upside down to remove the culture solution in the holes, each hole, add 150µl DMSO (Dimethyl sulfoxide), gently shake with a shaker for about 10 minutes in the dark at room temperature to completely dissolve the precipitate, use an automatic ELISA reader with a wavelength of 570-630nmnddeer to measure the OD value of composition in the pores, calculate the inhibition ratio.

Inhibition rate (%) = (1 – OD value of experimental group/OD value of comparison group) x 100

Calculate the IC50 number (Inhibitory concentration 50%)

3. Experiments on animals

3.1. Effect of Kacimex on experimental liver carcinogenesis (H22) in mice

3.1.1. Establishing a tumor-bearing mouse model

- Select 50 white mice, average weight 18-22g, half of each type male and female,

- With aseptic technique, using the acclimatization method in cancer, aspirating peritoneal fluid from liver cancer mice (H22) grown for 7 days, using physiological saline to mix into a cell suspension reaching a concentration of 3x107 cells. cells/ml.

- Use this cell suspension to inject subcutaneously into the armpit area of ​​experimental mice, injecting 0.2ml per animal.

- The next day, weigh the mouse's weight, kill the mouse, dissect the tumor, and weigh the tumor.

- Calculate the tumor growth inhibition rate according to the formula: Inhibition rate = (1 – average tumor weight in the experimental mouse group/average tumor weight in the control mouse group) x 100

3.1.2. Grouping and giving medication

- After being inoculated with H22 cancer cells above, mice were randomly divided into 5 groups after 24 hours: model group, Cyclophosphamide group, Kacimex group (high dose, medium dose and low dose).

- Model group: inject 0.4ml of warmed distilled water into the stomach, once a day, continuously for 10 days.

- The group used 0.2ml intraperitoneal injection of Cyclophosphamide to get a dose of 20mg/kg mouse body weight, once a day continuously for 10 days.

- Kacimex group (high dose, medium dose and low dose): gastric pump 0.4ml of Kacimex drug prepared into a differentiated solution reaching 40, 30, 15mg/ml according to different dose groups continuously for 10 days .

3.1.3. Determine the tumor inhibition rate of Kacimex in experimental animals

24 hours after the last drug administration, kill the mouse, dissect the tumor, and weigh the tumor.

Calculate the tumor inhibition rate according to the formula: Inhibition rate = (Average tumor weight in the model mouse group - Average tumor weight in the drug-treated mice group)/Average tumor weight in the group of model mice x 100

3.2. Effect of Kacimex drug on prolonging survival time of white mice with liver cancer line H22

3.2.1. Establishing a mouse model of H22 liver cancer with peritoneal effusion

- Select 50 white mice, average weight 18-22g, half of each type male and female,

- With aseptic technique, using the acclimatization method in cancer, aspirating peritoneal fluid from liver cancer mice (H22) grown for 7 days, using physiological saline to mix into a cell suspension reaching a concentration of 2x107 cells. cells/ml.

- Use this cell suspension to inject into the peritoneal cavity of experimental mice, injecting 0.2ml per animal.

3.2.2. Grouping and giving medication

 - After being implanted with the above H22 cancer cell lines, mice were randomly divided into 5 groups: model group, Cyclophosphamide group, Kacimex group (high dose, medium dose and low dose).

- Model group: inject 0.4ml of distilled water into the stomach, once a day, continuously for 10 days.

- The group used Cyclophosphamide injected subcutaneously with 0.2ml to get a dose of 20mg/kg of mouse body weight, once a day continuously for 10 days.

- Kacimex group (high dose, medium dose and low dose): gastric pump 0.4ml of Kacimex drug prepared into a differentiated solution reaching 40, 30, 15mg/ml according to different dose groups continuously for 10 days .

3.2.3. Observe the effects of Kacimex on the survival time of laboratory mice

After continuously giving the drug for 10 days, stop taking the drug and monitor the survival time of the experimental mice. Calculate the survival rate of experimental mice according to the following formula:

Rate of survival prolongation (%) = (Average number of survival days of the drug groups/average number of days of survival of the control group – 1) x 100%

RESULT

1. Results of in vivo experiments

Table 1. The inhibitory effect of Kacimex on cell proliferation is very clear and better than the comparison group.

Table 2. Inhibition effect of Kacimex on cell proliferation in people with liver cancer: Kacimex acts on HEPG2 cells in people with liver cancer within 48 hours, inhibiting the growth of HFPG2 cells not obvious.

Table 3. Effect of inhibiting cell proliferation of SGC - 7901 cells on people with stomach cancer.

Kacimex acts on SGC - 7901 cells in human gastric cancer within 48 hours, has the ability to inhibit cells quite clearly, and shows a dose-dependent relationship.

2. Results of in vivo experiments

Table 1. Effects of Kacimex during life on mice with abdominal cancer with water distention

Through the concentrations of the Kacimex group on the survival period of tumor mice, there was no obvious effect.

Table 2. Effect of Kacimex on tumor inhibition rate in mice

The weight of the Kacimex groups after treating tumor mice increased to different degrees, but the Kacimex treatment group with a low dose made the weight gain significantly higher than the Cyclophosphamide group.

CONCLUDE:

+ From experimental research results, it shows that:

1.Kacimex of the Vietnam Institute for Research and Treatment of Serious Diseases is produced from a number of herbs that help prevent and treat cancer at the cellular level.

2. Compared to the control group, Kacimex has the effect of inhibiting cell proliferation in experimental models. Thus, Kacimex has a good effect in treating liver cancer, stomach cancer and has the ability to prolong the life of people with the above dangerous diseases.

3. The difference between Kacimex and the control group is that in addition to the tumor treatment effect, there is no difference, Kacimex is non-toxic, does not cause unfavorable side effects, and also helps the body restore health.